We are indebted to the Leptospira collection of the Department of Microbiology and Immunology, Faculty of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico. We are grateful for the generous donation of the reference Leptospira strains; Leptospira fainei serovar Hurstbridge strain BUT6 and Leptospira biflexa serovar Patoc strain Patoc I to Dr. Alejandro de la Pe&#241;a Moctezuma. We thank Dr. Jos&#233; Antonio Ocampo Cervantes, the CIBAC Coordinator, and the personnel for their logistical support. EDT was under the Terminal Project program for undergraduate students of the Metropolitan Autonomous University-Campus Cuajimalpa. We acknowledge the Biorender.com software for the creation of figures 1, and 3&#160;to 9.

1. Sample preparation
<ol>
	<li>Concentrate each water sample in 1.5 mL microcentrifuge tubes by centrifugation at 8,000 x g for 10 min at 4 &#176;C. Repeat this step, as many times as required, to concentrate the sample into a volume of 250 &#181;L.</li>
	<li>Use the DNA extraction kit according to the manufacturer&#180;s instructions (see Table of Materials).</li>
	<li>Perform the specific PCR according to the dot-blot probe that will be used (Table 1).
	<ol>
		<li>Perform the amplifications in PCR tubes with the final volume of 25 &#181;L containing 1 X&#160;buffer, 2.5 units of Taq polymerase, 1 &#181;M of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl2, and 100 ng of target DNA from each water sample or reference genomic-DNA.</li>
		<li>Program the PCR reaction in a thermocycler according to the thermocycling conditions (Table 2).</li>
		<li>Store the reactions at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Place 10 &#181;L of each PCR product to be blotted in a separate well of a 96-well plate.</li>
	<li>Add 40 &#181;L of TE to each well and mix by pipetting. 
	NOTE: The 96-well plate can be sealed and stored at 4 &#176;C overnight. Follow the instructions provided in Supplementary File 6 to prepare buffers and solutions for the protocol. Figure 3 depicts the steps of sample preparation.</li>
</ol>
Table 2: PCR thermocycling conditions for the 16S, lipL41, and lipL32 genes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/TABLE_2_8th_PR_EMCC.xlsx" target="_blank">Please click here to download this Table.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65435/65435fig03.jpg" /> 
Figure 3: PCR and sample preparation. Applying the specific PCR protocol, the PCR product was transferred to a microtiter plate, and 40 &#181;L of TE was added to each well. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Assembling the dot-blot apparatus
NOTE: The assembling of the dot-blot apparatus is shown in Figure 4. During the procedure, wear gloves to handle the alkali solutions and protect the nylon membrane from contamination.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65435/65435fig04.jpg" /> 
Figure 4: Dot-blot apparatus assembling. The filter paper and the nylon membrane (previously moistened in 10 X&#160;SSPE) must be arranged in the correct order. The assembly must be secured with the screws tightly before applying the vacuum. Each well needs to be washed with TE, and the PCR products are loaded into their respective wells. After transferring the PCR product through the membrane, each well is washed again with TE and allowed to dry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Cut the nylon membrane (see Table of Materials) and filter paper into sheets of size 12&#160;x 8.5 cm.</li>
	<li>Mark the membrane with a permanent marker.</li>
	<li>Make a notch with scissors at one edge to indicate the right orientation. Use the mark to remember the sample order.</li>
	<li>Moisten the membrane and filter paper with 10 X Saline-sodium phosphate-EDTA buffer (SSPE; 3 M NaCl, 0.2 M NaH2PO4, and 0.02 M EDTA, pH 7.4) (Supplementary File 6). Handle the membrane with a clean, blunt-ended tweezer.</li>
	<li>Assemble the dot-blot chamber; first, the filter paper, followed by the membrane over&#160;the plastic seal. Secure the cover with the screws in a crosswise manner.</li>
	<li>Connect the chamber to the vacuum, place 100 &#181;L of TE in each well, hold the vacuum for 1 min, and then stop it.</li>
	<li>Switch on the vacuum at low speed and load 50 &#181;L of each sample into the corresponding well on the membrane of the dot-blot apparatus (following the pre-decided membrane distribution). 
	NOTE: Homogenize each sample in the hemagglutination 96 well plate prior to placing it in the dot-blot chamber.</li>
	<li>Allow the vacuum to dry the membrane. If necessary, hit the chamber gently to release the bubbles in the sample.</li>
	<li>Wash each well by placing 100 &#181;L of TE into it with a continuous vacuum and allowing it to dry. 
	NOTE: After completing the transfer, it is crucial to first switch off the pump and detach it. Failure to do so may result in reflux entering the apparatus. DNA does not need a denaturation step if a positively charged nylon membrane is used, but if using other inert support, a pre-denaturation step and alkali-based denaturation may be needed41.</li>
</ol>
3. DNA denaturation and fixation
NOTE: Figure 5 illustrates the DNA membrane fixation procedure.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65435/65435fig05v2.jpg" /> 
Figure 5: DNA-membrane fixation procedure. The DNA is denatured in an alkaline solution. Next, it is neutralized with 10 X&#160;SSPE, and the membrane is dried. Next, the membrane is transilluminated. The membrane is rehydrated with 2 X&#160;SSPE and pre-hybridized overnight. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig05largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Incubate the membrane with 0.4 M NaOH for 10 min at room temperature.</li>
	<li>Equilibrate the membrane with 10 X&#160;SSPE for 10 min at room temperature.</li>
	<li>Dry the membrane at 80 &#176;C for 2 h.</li>
	<li>Transilluminate with 120 mJ UV light for 3 min. Ensure that the samples are oriented face down towards the UV light source. If using a UV-crosslinker, confirm that the samples are facing upward and repeat this step twice. 
	NOTE: The membrane must be completely dry before UV crosslinking. DNA will be immobilized by a covalent bond to the nylon membrane. Each cross-linking takes about 18&#39;&#39; to 1&#39; with an optimal UV irradiation dose, for most hybridization experiments, of approximately 0.6-0.8 kJ/m2. 
	Exposure to UV irradiation is harmful to the eyes and skin. Wear appropriate protective equipment and avoid exposure to bare skin.</li>
	<li>Wash the membrane with 2&#160;X&#160;SSPE for 10 min.</li>
	<li>Carefully fold the membrane and insert it into the hybridization tube (use a 15 mL microcentrifuge tube).</li>
	<li>Add 10 mL of the pre-hybridization solution (Supplementary File 6) to the tube and incubate at 42 &#176;C overnight. Ensure that the tube has been properly sealed to prevent any leakage.</li>
</ol>
4. Hybridization
<ol>
	<li>Remove 5 mL of the pre-hybridization solution from the hybridization tube. To use the 5 mL for a second time, keep it in another tube and store it at -4 &#176;C.</li>
	<li>Add 15 &#181;L of the labeled probe (Table 1) to the hybridization tube. Rinse the tip into the solution to ensure that the labeled probe was thoroughly instilled into the dilution. 
	NOTE: The DNA probe&#39;s binding is not as strong as the antigen-antibody binding. Therefore, changes in the concentration of the probe or DNA in the sample can influence the intensity of the emitted fluorescence. DNA probes are stoichiometric, meaning that the number of probe molecules bound to DNA is equivalent to the number of DNA molecules present in the solution. Figure 6 depicts the probe hybridization.</li>
	<li>Incubate at 42 &#176;C overnight.</li>
	<li>Perform DNA probe DIG labeling. Follow the instructions specified in Table 3 to mix the reagents, then incubate the mixture at 37 &#176;C for 1 h. Next, add 80 &#181;L of distilled water and store the tailed probe at 4 &#176;C.</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65435/65435fig06.jpg" /> 
Figure 6: Probe hybridization. The volume of the hybridization buffer is adjusted, and the digoxigenin-labeled probe is incorporated to allow the probe&#39;s hybridization overnight. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 3: Reagents for the probes labeling with digoxigenin (DIG). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/TABLE_3_EMCC_V4.zip" target="_blank">Please click here to download this Table.</a>
5. Chemiluminescence (anti-DIG tagging)
<ol>
	<li>Wash the membrane twice with 2&#160;X&#160;SSPE/0.1% SDS at room temperature for 10 min.</li>
	<li>Incubate the membrane with 5&#160;X&#160;SSPE/0.1% SDS at the probe&#39;s annealing temperature for 15 min. 
	NOTE: In this step, use a container with a lid to maintain the temperature as long as possible. Ensure uniform and constant temperature across the membrane. The approximate volume in each wash is 100 mL of the solution&#160;indicated.</li>
	<li>Wash the membrane once with Buffer 1 (Supplementary File 6) at room temperature for 5 min.</li>
	<li>Wash the membrane once with Buffer 2 (Supplementary File 6) at room temperature for 30 min.</li>
	<li>Wash the membrane with Buffer 2 and add the anti-digoxigenin antibody (15 &#181;L) (see Table of Materials), then incubate for 45 min (the incubation time may vary from 30-60 min). This secondary antibody tags the probe for detection. 
	NOTE: The membrane may be stored in Buffer 2 with the anti-digoxigenin antibody overnight at 4-8 &#176;C. Anti-DIG tagging of the chemiluminescence process is depicted in Figure 7.</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65435/65435fig07.jpg" /> 
Figure 7: Anti-DIG tagging of the chemiluminescence process. The unbonded nucleic acids are removed with buffer solutions. The probe is aligned with the target DNA, and the excess is removed. The membrane is blocked with the blocking 1&#160;X&#160;buffer, and the anti-DIG antibody is added (1:10000). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Chemiluminescence (substrate application)
<ol>
	<li>Wash the membrane twice in Buffer 1 at room temperature for 15 min.</li>
	<li>Wash once in Buffer 3 (see Supplementary file 6) at room temperature for 5 min, allowing the membrane to drain most of the buffer, leaving it almost dry.</li>
	<li>Add CSPD ready to use (Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2&#8242;-(5&#8242;-chloro) tricyclo [3.3.1.13,7] decan}-4-yl) phenyl phosphate, see Table of Materials), and let stay for 5 min, (with damped fingers homogenize the CSPD from one side to the other).</li>
	<li>Place the membrane in a transparent plastic bag (12.5&#160;x 9 cm) that fits the membrane dimension. Carefully remove air bubbles, evenly distribute the CSPD, and heat-seal the bag.</li>
	<li>Incubate the membrane in a water bath at 37 &#176;C for 15 min (it can be up to 30 min), and ensure that the membrane with samples is placed downwards so that it is well submerged. Ensure that the temperature remains constant and well distributed over the membrane.</li>
	<li>Dry the plastic bag and fix it with scotch tape on an already-used radiographic film. This will allow easy handling during the exposure procedure. 
	NOTE: Figure 8 shows the substrate application of the chemiluminescence process.</li>
</ol>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65435/65435fig08v2.jpg" /> 
Figure 8: Substrate application of the chemiluminescence process. The free antibody is removed, and the substrate CSPD (1:250) is added to the membrane. The reaction is activated by incubation at 37 &#176;C and the membrane is arranged to record the chemiluminescence in an X-ray film. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig08largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. Chemiluminescence (detection)
<ol>
	<li>Perform the exposure procedure. In the darkroom, prepare the developer and fixer solutions (see Table of Materials) in the respective trays.</li>
	<li>In the dark, carefully place the fixed membrane facing a new radiographic film&#160;and insert it into a radiographic cassette (see Table of Materials).</li>
	<li>Register the time of exposure. The time of exposure may vary from 1 min to 30 min or more. Start with 5 min and adjust the time accordingly.</li>
	<li>Damp the X-ray film in developer solution for 1-3 min and rinse gently with tap water (15-45 s).</li>
	<li>Damp the X-ray film in fixer solution for 1-3 min and rinse gently with tap water (45 s).</li>
	<li>Allow the X-ray film to air dry and document the assay in a visible white lightbox. 
	NOTE: Avoid shaking the membrane during the X-ray exposure time. The detection process is depicted in Figure 9.</li>
	<li>Proceed to the result interpretation. In the exposed X-ray film, the dots with emission can be visually located and correspond to the DNA fragments with the probe&#39;s hybridization. The intensity of the emitted signal depends on the luminescence and the duration of the exposure. 
	NOTE: Membranes can be stored dried between filter paper for several months at room temperature.</li>
</ol>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65435/65435fig09.jpg" /> 
Figure 9: Detection of the chemiluminescence process. Under dark conditions, the membrane is exposed to an X-ray film inside an X-ray cassette. Next, it was allowed to stand during the exposition time, and then the X-ray film was developed and fixed. Finally, it was air dried and interpreted. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
8. Membrane de-hybridization procedure
<ol>
	<li>Wash the membrane twice for 10 min with distilled water.</li>
	<li>Wash the membrane for 20 min with 0.4 M NaOH at 53 &#176;C (twice).</li>
	<li>Wash twice the membrane for 10 min with 2 X SSPE.</li>
	<li>Allow the membrane to dry at room temperature.</li>
	<li>Keep the membrane and return to step 3.5 of the protocol. 
	NOTE: The membrane may be stored in the pre-hybridization buffer overnight at 4-8 &#176;C. The next day, restart with an incubation at 42 &#176;C for 1 h, allowing the pre-hybridization buffer to reach the temperature. Then, proceed to step 3.5 of the protocol.</li>
</ol>

Dot-Blot Assay for Detection of Leptospira DNA in Water Samples

The authors declare that there is no conflict of interest.

The critical steps of the dot-blot technique include (1) DNA immobilization, (2) blocking of the free binding sites on the membrane with non-homologous DNA, (3) the complementarity between the probe and the target fragment under annealing conditions, (4) removal of the unhybridized probe, and (5) the detection of the reporter molecule41.
The PCR-Dot-blot has certain limitations, such as the technique does not provide information about the size of the hybridized fragment37, it requires de-hybridization of a single membrane before re-hybridization with a second or third probe, and it requires considerable time and effort to the final result.
This technique has shown consistency with other molecular techniques42,43 and its applications. Reverse dot-blot, nanogold dot-blot21, and dot-ELISA42 have been employed for the detection of specific genes in organisms like Treponema44, Borrelia burgdorferi45, E. coli46, Helicobacter pylori47, and Mycobacterium tuberculosis48,49 based on the 16S&#160;rRNA29,43, and for the detection of DNA or RNA of plant viruses41.&#160;The limit of detection (LOD) defined as the lowest concentration of the microorganism that can be consistently and reliably measured by this technique50, ranged from 100 pg (5 x 104 bacteria) for H. pylori47, to a single cell of B. burgdorferi in clinical samples45, and a fg, which is less than a cell, of M. tuberculosis48,49.
One valuable aspect of this combined methodology is its ability to extend the detection limit by one order, overcoming false negative results due to two sample conditions that may lead to inconsistent results: (1) samples with insufficient DNA, or (2) sample contamination, allowing 100 % detection of positive samples25,45,47,48,49. The PCR-Dot-blot has the capacity to detect lower quantities of amplified products than those that can be detected in agarose gel electrophoresis, and it avoids contamination as a consequence of second amplification steps, as in nested and semi-nested PCR. These advantages are particularly critical when working with environmental samples containing a low bacterial charge, which may contain a broader range of inhibitors and DNA from unrelated sources that may interfere with the molecular techniques.
Previous work suggests that leptospires may be identified by DNA probes labeled with photobiotin reaching a LOD of 5 pg (5 x 103 leptospires)51. DIG-labeled probes detect 0.1-1 pg (102&#160;leptospires), while 32P-labeled probes detect 1-5 pg (750 to 1000 leptospires)51, and biotin-labeled probes detect 5 pg (2500 leptospires)52. Further, 32P-labeled probes for saprophyte Leptospira reached a&#160;LOD of 1.95 ng and for pathogenic Leptospira, 3.9 ng53.&#160;In this study, similar LODs were established for the three clades. The LOD for pathogenic species was approximately 0.3 ng of genomic DNA (6-8 x 104 GEq) (Figure 11), and for the saprophyte and intermediate species, it was down to 0.1 ng (2 x 104 GEq). Notably, previous researches were conducted using pure Leptospira cultures51 and sera from experimentally infected golden hamsters, aiming to detect pathogenic Leptospira54,55, and to apply DNA hybridization with 32P labeled as a tool for the identification and classification of Leptospira56. These probes exhibit high specificity and do not show cross-hybridization with non-homologous DNA from other microorganisms19,51,54,55,56,57, and can distinguish even among closely related species such as Leptonema57. Likewise, in this work, no hybridization with non-homologous DNA was observed. Although non-isotopic probes are not as sensitive as isotopic probes58 or other techniques43, it can be expected that the PCR-Dot-blot combination allows up to a tenfold increase in sensitivity compared to other PCR-based techniques, as seen in the detection of Leishmania and the bovine herpesvirus 4 (BHV4)24,26.
On the other hand, the dot-blot assay for Leptospira identification has been more frequently employed with antibodies, and&#160;lately&#160;with monoclonal antibodies as an alternative for diagnosing humans and animals in urine samples59,60,61,62. In these studies, the sample is directly processed onto the membrane without prior steps, and antibodies target specific Leptospira surface proteins associated with pathogenesis. Mab-Dot-blot ELISA, for example, shows high sensitivity and specificity, detecting as little as 1 &#181;g of bacterial homogenate62. The results obtained from the Mab-Dot-blot assay can be comparable to PCR assays, which typically have LOD around 103 leptospires/ml of urine or 9.3 ng of Leptospira homogenate63. The amount of genomic DNA that can be amplified by PCR is 100 cells (500 fg of DNA)61. These previous studies used pure cultures or clinical samples without enrichment procedures and were oriented toward better diagnosis. Given that infected rats can excrete as many as 107 leptospires/mL of urine64, and dogs can excrete between 102 to 106 leptospires/mL of urine65, serving as sources of contamination for water bodies, the environmental samples require leptospiral enrichment. The in vitro PCR amplification of the targeted DNA before blotting allows for an improvement in procedure sensitivity of up to ten-fold52,55. Usually, the product amplified by an end-point PCR would not be visualized in an agarose gel electrophoresis; nonetheless, it becomes evident through the specific hybridization of the DIG-labeled probe in the dot-blot.
While much research on Leptospira and leptospirosis has focused on disease diagnosis, little is known about their ecology in water bodies and their distribution within them. The PCR-Dot-blot technique adds to the toolbox for studying Leptospira and leptospirosis, offering the advantage of screening a large number of samples and expediting results and interpretation. PCR-Dot-blot is a relatively low-cost, simple, and non-radioactive technique that can accommodate multiple probes on a single membrane. Besides, it eliminates the need for sophisticated equipment and complex workflows, which can be challenging in resource-limited settings.
The impact of this study lies in its future application to study large bodies of water at different depths, as well as other ecological niches and sample types, including soil, stagnant water, feces, blood, and tissues.
In conclusion, this work demonstrates the application of the PCR-Dot-blot technique, based on the digoxigenin labeling of DNA probes for the identification of the three clades within the genus Leptospira. This method streamlines the identification process by reducing it to a single amplification step and allows for either three simultaneous hybridizations or three consecutive hybridizations using a single membrane. The detection limits achieved with this technique are similar to those achieved with radioactive probes53, making it a valuable tool for identifying Leptospira in water samples from natural sources. Consequently, this technique offers increased sensitivity and provides accurate and consistent results when evaluating samples with minimal amounts of bacterial DNA. It serves as an alternative approach for studying the dynamics of Leptospira in water bodies and may aid the assessment and surveillance of broader environmental samples, aiding in the implementation of preventive measures against the transmission of this microorganism.
In case of difficulties with the technique, consider the following troubleshooting highlights: (1) If the dots have irregular shapes, check that the vacuum system is securely closed crosswise and then repeat the transfer. (2) In case the hybridization tube breaks during the overnight procedure, causing the membrane to lose the hybridization buffer, refer to the &#34;De-hybridization procedure&#34; section and restart the process. (3) If no dots are visible, do not discard the membrane; proceed to the &#34;De-hybridization procedure&#34; section to repeat the hybridization steps. (3) If uneven white areas are noticed on the developed X-ray film, ensure that no air bubbles are trapped during the bag-sealing process. (5) If uneven black areas are observed on the developed X-ray film, confirm that the CSPD has been thoroughly distributed using damped fingers. (6) If the X-ray film shows shadows of the dots, ensure that the film is not moved during the exposure time.

Leptospirosis in humans mainly originates from environmental sources1,2. The presence of Leptospira in lakes, rivers, and streams is an indicator of leptospirosis transmission among wildlife, and domestic and production animals that may eventually come into contact with these bodies of water1,3,4. Furthermore, Leptospira has been identified in non-natural sources, including sewage, stagnant and tap water5,6.
Leptospira is a worldwide distributed bacteria7,8, and the role of the environment in its preservation and transmission has been well recognized. Leptospira can survive in drinking water under variable pH and minerals9, and in natural bodies of water1. It can also survive for long periods in distilled water10, and under constant pH (7.8), it may survive up to 152 days11. Moreover, Leptospira may interact in bacterial consortiums to survive harsh conditions12,13.&#160;It may be part of biofilms in freshwater with Azospirillum and Sphingomonas and is even capable of growing and enduring temperatures exceeding 49 &#176;C14,15. It can also multiply in waterlogged soil and remain viable for up to 379 days16, preserving its ability to cause the disease for as long as a year17,18. However, little is known about the ecology within water bodies and how it is distributed within them.
Since its discovery, the study of the genus Leptospira was based on serological tests. It was not until the current century that molecular techniques became more prevalent in the study of this spirochaete. The dot-blot has been scarcely used for its identification using (1) an isotopic probe based on the 16S rRNA and on an inter-simple sequence repeat (ISSR)19,20, (2) as a nanogold based immunoassay for human leptospirosis applied to urine21, or (3) as an antibody-based assay for bovine urine samples22. The technique fell in disuse because it was originally based on isotopic probes. However, it is a well-known technique that, coupled with PCR, yields enhanced results, and it is considered safe due to the use of non-isotopic probes. PCR plays a crucial role in the enrichment of the Leptospira DNA by amplifying a specific DNA fragment that may be found in trace amounts in a sample. During each PCR cycle, the amount of the targeted DNA fragment is doubled in the reaction. At the end of the reaction, the amplicon has been multiplied by a factor of more than a million23. The product amplified by PCR, often not visible in agarose electrophoresis, becomes visible through specific hybridization with a DIG-labeled probe in the dot-blot24,25,26.
The dot-blot technique is simple, robust, and suitable for numerous samples, making it accessible to laboratories with limited resources. It has been employed in a variety of bacteria studies, including (1) oral bacteria27, (2) other sample types such as food and feces28, and (3) the identification of unculturable bacteria29, often in agreement with other molecular techniques. Among the advantages offered by the dot-blot technique are: (1) The membrane has a high binding capacity, capable of binding over 200 &#956;g/cm2 of nucleic acids and up to 400 &#956;g/cm2; (2) Dot-blot results can be visually interpreted without requiring special equipment, and (3) they can be conveniently stored for years at room temperature (RT).
The genus Leptospira has been classified into pathogenic, intermediate, and saprophytic clades30,31. The distinction among these clades can be achieved based on specific genes such as lipL41, lipL32, and the 16S&#160;rRNA. LipL32 is present in the pathogenic clades and exhibits high sensitivity in various serological and molecular tools, whereas it is absent in saprophyte species21. The housekeeping gene lipL41 is&#160;known for its stable expression and used in molecular techniques32, while the 16S&#160;rRNA gene is utilized for their classification.
This methodology can be applied to large volumes of water once they have been concentrated by centrifugation. It allows the assessment of various points and depths within a water body to detect the presence of leptospiral DNA and the clade to which it belongs. This tool is valuable for both ecological and general screening purposes and can also be employed to detect other non-culturable bacteria that may be present in water.
Additionally, PCR and dot-blot assays are technically and economically affordable to a wide range of laboratories, even those lacking sophisticated or expensive equipment. This study aims to apply the digoxigenin-based dot-blot for the identification of the three Leptospira clades in water samples collected from natural bodies of water.
Bacterial strains 
Twelve Leptospira serovars (Autumnalis, Bataviae, Bratislava, Canicola, Celledoni, Grippothyphosa, Hardjoprajitno, Icterohaemorrhagiae, Pomona, Pyrogenes, Tarassovi, and Wolffi) were included in this study. These serovars are part of the collection at the Department of Microbiology and Immunology, Faculty of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico,&#160;and they are currently used in the microagglutination test (MAT).
All Leptospira serovars were cultured in EMJH, and their DNA was extracted using a commercial DNA extraction kit (see Table of Materials). A genomic DNA mix of the twelve serovars was used as a positive control for the Leptospira pathogenic clade. As a positive control of the Leptospira intermediate clade, genomic DNA from Leptospira fainei serovar Hurstbridge strain BUT6 was included, and as a positive control for the Leptospira saprophyte clade, genomic DNA of Leptospira biflexa serovar Patoc strain Patoc I, was also included.
Negative controls consisted of an empty plasmid, DNA from non-related bacteria (Ureaplasma urealyticum, Staphylococcus aureus, Brucella abortus, Salmonella typhimurium, Shigella boydii, Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli), and PCR-grade water, which served as non-template control.
Water samples 
Twelve trial grab samples were collected using a stratified-haphazard sampling method from the Cuemanco Biological and Aquaculture Research Center (CIBAC) (19&#176; 16&#39; 54&#34; N 99&#176; 6&#39; 11&#34; W). These samples were obtained at three depths: superficial, 10, and 30 cm (Figure 1A, B). The water collection procedures did not impact any endangered or protected species. Each sample was collected in a sterile 15 mL microcentrifuge tube. To collect the sample, each tube was gently submerged in the water, filled at the selected depth, and then sealed. The samples were maintained at 22 &#176;C and promptly transported to the laboratory for processing.
Each sample was concentrated by centrifugation in sterile 1.5 mL microcentrifuge tubes at 8000 x g for 20 min at room temperature. This step was repeated until all the samples were concentrated into one tube, which was then used for DNA extraction (Figure 1C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65435/65435fig01v2.jpg" /> 
Figure 1: Concentration of water samples by centrifugation. (A) Water sampling ponds, and (B) Natural streams. (C) Centrifugation-based water sample processing in repeated steps as many times as needed (n). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
DNA extraction 
Total DNA was isolated using a commercial Genomic DNA kit according to the manufacturer&#39;s instructions (see Table of Materials). DNA extractions were eluted in 20 &#181;L of elution buffer, and DNA concentration was determined by a UV Spectrophotometer at 260-280 nm, and stored at 4 &#176;C until use.
PCR amplification 
The PCR targets were the 16S rRNA, lipL41, and lipL32 genes, which identify DNA from the genus Leptospira and allow the distinction among the three clades: pathogenic, saprophytic, and intermediate. Both primers and probe designs were based on the previous works by Ahmed et al., Azali et al., Bourhy et al., Weiss et al., and Branger et al.33,34,35,36,37. The sequence of each probe, primer, and amplified fragment are described in Table 1, and their alignment with reference sequences are provided in Supplementary File 1, Supplementary File 2, Supplementary File 3, Supplementary File 4, and Supplementary File 5. The PCR reagents and thermocycling conditions are described in the protocol section.
Amplification products were visualized by electrophoretic separation on a 1% agarose gel in TAE (40 mM Tris base, 20 mM Acetic acid, and 1 mM EDTA; pH 8.3), at 60 V for 45 min with ethidium-bromide detection, as shown in Supplementary Figure 1. Genomic DNA obtained from each serovar was used with concentrations ranging from 6 x 106 to 1 x 104 genomic equivalent copies (GEq) in each PCR reaction, based on the genome size of L. interrogans (4, 691, 184 bp)38 for pathogenic Leptospira, the genome size of L. biflexa (3, 956, 088 bp)39 for saprophytic Leptospira, and the genome size of L. fainei serovar Hurstbridge strain BUT6 (4, 267, 324 bp) with accession number AKWZ00000000.2.
The sensitivity of the probes was assessed with DNA from each pathogenic serovar, L. biflexa serovar Patoc strain Patoc I, and L. fainei serovar Hurstbridge strain BUT6 in each experiment. To assess the specificity of the PCR and dot-blot hybridization assay, DNA from non-related bacteria was included.
Table 1: PCR primers and probes to amplify products for identifying the pathogenic, saprophyte, and intermediate clades of Leptospira. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/TABLE_1_ 7th_PR_ EMCC.xlsx" target="_blank">Please click here to download this Table.</a>
Dot-blot hybridization assay 
The technique is called dot-blot because the holes in which the DNA sample is placed have a dot shape, and when they are sucked to be fixed in place by vacuum suction, they acquire this shape. This technique was developed by Kafatos et al.40. The technique allows the semi-quantification of Leptospira in each PCR-positive sample. The protocol consists of a denaturation with NaOH 0.4 M at room temperature, samples with Leptospira DNA from 30 ng to 0.05 ng, corresponding to 6 x 106 to 1 x 104 leptospires, are blotted onto a nylon membrane with a 96-well dot-blot apparatus. After immobilization, the DNA is bound to the membrane by exposure to 120 mJ UV light. Each DNA probe is conjugated with digoxigenin-11 dUTP by a terminal transferase catalysis step at the 3&#39; end (Digoxigenin is a plant steroid obtained from Digitalis purpurea, used&#160;as a reporter41). Following the stringent hybridization of the labeled DNA probe (50 pmol) at the specific temperature onto the target DNA, the DNA hybrids are visualized by the chemiluminescence reaction with the anti-digoxigenin alkaline phosphatase antibody covalently conjugated with its substrate CSPD. The luminescence is captured by exposure to an X-ray film (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65435/65435fig02v2.jpg" /> 
Figure 2: Steps of the procedure for the PCR-dot-blot assay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Purelink DNA extraction kit</td><td>Invitrogen</td><td>K182002</td><td></td></tr><tr><td>Gotaq Flexi DNA Polimerase (End-Point PCR Taq polymerase kit)</td><td>Promega</td><td>M3001</td><td></td></tr><tr><td>Whatman filter paper, grade 1,</td><td>Merk</td><td>WHA1001325</td><td></td></tr><tr><td>Nylon Membranes, positively charged Roll 30cm x 3 m</td><td>Roche</td><td>11417240001</td><td></td></tr><tr><td>Anti-Digoxigenin-AP, Fab fragments Sheep Polyclonal Primary-antibody</td><td>Roche</td><td>11093274910</td><td></td></tr><tr><td>Medium Base EMJH</td><td>Difco</td><td>S1368JAA</td><td></td></tr><tr><td>Leptospira Enrichment EMJH</td><td>Difco</td><td>BD 279510</td><td></td></tr><tr><td>Blocking Reagent</td><td>Roche</td><td>11096176001</td><td></td></tr><tr><td>CSPD ready to use Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2&amp;prime;-(5&amp;prime;-chloro) tricyclo [3.3.1.1&lt;sup&gt;3,7&lt;/sup&gt;] decan}&lt;sup&gt;8&lt;/sup&gt;-4-yl) phenyl phosphate</td><td>Merk</td><td>11755633001</td><td></td></tr><tr><td>Deoxyribonucleic acid from herring sperm</td><td>Sigma Aldrich</td><td>D3159</td><td></td></tr><tr><td>Developer Carestream</td><td>Carestream Health Inc</td><td>GBX5158621</td><td></td></tr><tr><td>Digoxigenin-11-ddUTP</td><td>Roche</td><td>11363905910</td><td></td></tr><tr><td>EDTA, Disodium Salt (Dihydrate)</td><td>Promega</td><td>H5032</td><td></td></tr><tr><td>Ficoll 400</td><td>Sigma Aldrich</td><td>F8016</td><td></td></tr><tr><td>Fixer Carestream</td><td>Carestream Health Inc</td><td>GBX 5158605</td><td></td></tr><tr><td>Lauryl sulfate Sodium Salt (Sodium dodecyl sulfate; SDS) C&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;25&lt;/sub&gt;0&lt;sub&gt;4&lt;/sub&gt;SNa</td><td>Sigma Aldrich</td><td>L5750</td><td></td></tr><tr><td>N- Lauroylsarcosine sodium salt CH&lt;sub&gt;3&lt;/sub&gt;(CH&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;10&lt;/sub&gt;CON(CH&lt;sub&gt;3&lt;/sub&gt;) CH&lt;sub&gt;2&lt;/sub&gt;COONa</td><td>Sigma Aldrich</td><td>L-9150</td><td>It is an anionic surfactant</td></tr><tr><td>Polivinylpyrrolidone (PVP-40)</td><td>Sigma Aldrich</td><td>PVP40</td><td></td></tr><tr><td>Polyethylene glycol Sorbitan monolaurate (Tween 20)</td><td>Sigma Aldrich</td><td>9005-64-5</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma Aldrich</td><td>151-21-3</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma Aldrich</td><td>1310-73-2</td><td></td></tr><tr><td>Sodium phosphate dibasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4)&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7558-79-4</td><td></td></tr><tr><td>Terminal transferase, recombinant</td><td>Roche</td><td>3289869103</td><td></td></tr><tr><td>Tris hydrochloride (Tris HCl)</td><td>Sigma-Aldrich</td><td>1185-53-1</td><td></td></tr><tr><td>SSPE 20X</td><td>Sigma-Aldrich</td><td>S2015-1L</td><td>It can be Home-made following Supplementary File 6</td></tr><tr><td>Primers</td><td>Sigma-Aldrich</td><td>On demand</td><td>Follow table 1</td></tr><tr><td>Probes</td><td>Sigma-Aldrich</td><td>On demand</td><td>Follow table 1</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nanodrop&amp;trade; One Spectrophotometer</td><td>Thermo-Scientific</td><td>ND-ONE-W</td><td></td></tr><tr><td>Refrigerated microcentrifuge Sigma 1-14K, suitable for centrifugation of 1.5 ml microcentrifuge tubes at 14,000 rpm</td><td>Sigma-Aldrich</td><td>1-14K</td><td></td></tr><tr><td>Disinfected adjustable pipettes, range 2-20 &amp;micro;l, 20-200 &amp;micro;l</td><td>Gilson</td><td>SKU:F167360</td><td></td></tr><tr><td>Disposable 1.5 ml microcentrifuge tubes (autoclaved)</td><td>Axygen</td><td>MCT-150-SP</td><td></td></tr><tr><td>Disposable 600 &amp;micro;l microcentrifuge tubes (autoclaved)</td><td>Axygen</td><td>3208</td><td></td></tr><tr><td>Disposable Pipette tips 1-10 &amp;micro;l</td><td>Axygen</td><td>T-300</td><td></td></tr><tr><td>Disposable Pipette tips 1-200 &amp;micro;l</td><td>Axygen</td><td>TR-222-Y</td><td></td></tr><tr><td>Dot-Blot apparatus Bio-Dot</td><td>BIORAD</td><td>1706545</td><td></td></tr><tr><td>Portable Hergom Suction</td><td>Hergom</td><td>7E-A</td><td></td></tr><tr><td>Scientific Light Box (Visible-light PH90-115V)</td><td>Hoefer</td><td>PH90-115V</td><td></td></tr><tr><td>UV Crosslinker</td><td>Hoefer</td><td>UVC-500</td><td></td></tr><tr><td>Thermo Hybaid PCR Express Thermocycler</td><td>Hybaid</td><td>HBPX110</td><td></td></tr><tr><td>Radiographic cassette with IP Plate14 X 17</td><td>Fuji</td><td></td><td></td></tr></tbody></table>

polymerase chain reaction and dot-blot hybridization for leptospira detection in water samples

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe hybridization in the presence of carrier DNA. It is based on the transfer of a known amount of DNA onto an inert solid support, such as a nylon membrane, utilizing the dot-blot apparatus and without electrophoretic separation. Nylon membranes have the advantage of high nucleic acid binding capacity (400 &#181;g/cm2), high strength, and are positively or neutrally charged. The probe used is a highly specific ssDNA fragment of 18 to 20 bases long labeled with digoxigenin (DIG). The probe will conjugate with the Leptospira DNA. Once the probe has hybridized with the target DNA, it is detected by an anti-digoxigenin antibody, allowing its easy detection through its emissions revealed in an X-ray film. The dots with an emission will correspond to the DNA fragments of interest. This method employs the non-isotopic labeling of the probe, which may have a very long half-life. The drawback of this standard immuno-label is a lower sensitivity than isotopic probes. Nevertheless, it is mitigated by coupling polymerase chain reaction (PCR) and dot-blot assays. This approach enables the enrichment of the target sequence and its detection. Additionally, it may be used as a quantitative application when compared against a serial dilution of a well-known standard. A dot-blot application to detect Leptospira from the three main clades in water samples is presented here. This methodology can be applied to large amounts of water once they have been concentrated by centrifugation to provide evidence of the presence of Leptospiral DNA. This is a valuable and satisfactory tool for general screening purposes, and may be used for other non-culturable bacteria that may be present in water, enhancing the comprehension of the ecosystem.

To assess the effectiveness of the technique, genomic DNA from pure cultures of each Leptospira serovar was used, along with the clade-specific probe. Membranes were prepared with 100 ng of genomic DNA per PCR reaction for each serovar, followed by eight genomic DNA of non-related bacteria and variable concentrations of genomic DNA of the ad hoc Leptospira serovars. Each assay included positive, negative, and non-template control. These non-related genomic DNA did not show an affinity for the dot-blot probes. The membrane distribution and dot-blot membranes are shown in Supplementary Figure&#160;2,&#160;Supplementary Figure 3,&#160;Supplementary Figure 4,&#160;Supplementary Figure 5,&#160;Supplementary Figure 6,&#160;Supplementary Figure&#160;7. Regarding the saprophyte clade, using the DB_Saproprobe with the PCR product of 1059 bp amplified with primers DB_16SPathoREV and DB_16SPathoFWD, and the PCR product of 517 bp amplified with primers DB_16SSapREV and DB_16SSapFWD with genomic DNA of Leptospira biflexa serovar Patoc strain Patoc I, it was possible to detect between 0.05-0.1 ng of saprophyte DNA (Supplementary Figure 2 and Supplementary Figure 3). Likewise, for the detection of the intermediate clade, using the DB_Interprobe with the PCR product of 1059 bp amplified with primers DB_16SPathoREV and DB_16SPathoFWD, and the PCR product of 299-301 bp amplified with the primers DB_16SInterREV and DB_16SInterFWD with genomic DNA of Leptospira fainei serovar Hurstbridge strain BUT6 (Supplementary Figure 4 and Supplementary Figure 5), it was possible to detect around 0.1 ng of the intermediate DNA. Similarly, for the pathogenic clade using the DB_16SPathoprobe with the PCR product of 1059 bp amplified by primers DB_16SPathoREV and DB_16SPathoFWD, or the DBlipL41probe with the PCR product of 479 bp amplified by primers DB_lipL41REV and DB_lipL41FWD with genomic DNA mix from pathogenic Leptospira serovars, it was possible to detect DNA concentrations ranging 0.3-0.6 ng of pathogenic DNA (Supplementary Figure 6 and Supplementary Figure 7).
The assays designed to identify the three clades can be used individually for each clade. However, in the case of valuable and scarce samples, a single membrane may be prepared with primers DB_16SPathoREV and DB_16SPathoFWD, which amplify a 1058-1069 bp product. This product can then be hybridized with the DB_Interprobe, the DB_Saproprobe, and the DB_16SPathoprobe individually, with the de-hybridization step (step 8) before each one of them. This approach allows for the identification of all clades in the same membrane.
In the field water samples, the lipL32 probe protocol was applied. Each water sample&#39;s DNA was used at a concentration of 100 ng per PCR reaction. Additionally, a DNA extraction protocol control was included, which consisted of spiking 100 ng of Leptospira genomic DNA into a 15 mL distilled water sample. Negative and positive controls were also included (Figure 10).
Table 4: Water samples description and depth. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/TABLE_4_EMCC_R2.xlsx" target="_blank">Please click here to download this Table.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65435/65435fig10.jpg" /> 
Figure 10: Dot-blot assay of each water sample. (A) Membrane distribution and (B) dot-blot of the DB_lipL32probe and the product (262 bp) of primers DB_lipL32REV and DB_lipL32FWD of the field water samples. The surface of Pond 3 shows a positive result, and both the extraction protocol control and the positive controls have an intense signal. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The water sample description is shown in Table 4, and the dot-blot membrane distribution and image are shown in Figure 9. The dot A7 shows that Leptospiral DNA was present on the surface of pond 3. In order to determine the DNA concentration that was present in pond 3, a dot-blot assay with known DNA concentrations of a mix of Leptospira serovars and the lipL32 probe was performed (Figure 11). The membrane was prepared with genomic DNA distributed from 100 ng to 1 x 10-13 ng in each dot. The minimum concentration that can be undoubtedly detected was approximately 0.3 ng, corresponding to 6 x 105 GEq in the water samples.
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/65435/65435fig11.jpg" /> 
Figure 11: Dot-blot assay with known DNA concentrations of a mix of Leptospira serovars and the lipL32 probe. (A) Membrane distribution and (B) dot-blot of the DB_lipL32probe and the product (262 bp) of primers DB_lipL32REV and DB_lipL32FWD with dilutions (1:2) of a genomic DNA mix of pathogenic Leptospira serovars. The probe&#39;s signal can be easily detected down to 3.91 x 10-1 ng of Leptospira DNA mix. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/65435fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: PCR amplification products visualized by electrophoretic separation. Lane 1. Molecular weight marker 100 bp DNA Ladder. Lane 2. PCR based on the lipL32 gene with pathogenic Leptospira DNA mix as a template; Lane 3. PCR based on the lipL41 gene with pathogenic Leptospira DNA mix as a template; Lane 4. PCR based on the 16S rRNA gene with pathogenic Leptospira DNA mix as template; Lane 5. PCR based on the 16S rRNA gene with primers DB_16SSapREV and DB_16SSapFWD, and Leptospira biflexa serovar Patoc strain Patoc I DNA as template; Lane 6. PCR based on the 16S rRNA gene with primers DB_16SPathoREV and DB_16SPathoFWD, and Leptospira biflexa serovar Patoc strain Patoc I DNA as template; Lane 7. PCR based on the 16S rRNA gene with primers DB_16SInterREV and DB_16SInterFWD, and Leptospira fainei, serovar Hurstbridge strain BUT6 DNA as template; Lane 8. PCR based on the 16S rRNA gene with primers DB_16SPathoREV and DB_16SPathoFWD, and Leptospira fainei, serovar Hurstbridge strain BUT6 DNA as a template. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/Supp FIGURE_1_EMCC.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: (A) Membrane distribution and (B) dot-blot of the DB_Saproprobe and the product (1059 bp) of primers DB_16SPathoREV and DB_16SPathoFWD of genomic DNA of Leptospira biflexa serovar Patoc, Patoc I.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/Supp FIGURE_2_EMCC.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: (A) Membrane distribution and (B) dot-blot of the DB_Saproprobe with the product (517 bp) of primers DB_16SSapREV and DB_16SSapFWD of genomic DNA of Leptospira biflexa serovar Patoc, Patoc I.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/Supp FIGURE_3_EMCC.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4: (A) Membrane distribution and (B) dot-blot of the DB_Interprobe with the product (1059 bp) of primers DB_16SPathoREV and DB_16SPathoFWD and genomic DNA of Leptospira fainei serovar Hurstbridge strain BUT6.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/Supp FIGURE_4_5th_PR_EMCC.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 5: (A) Membrane distribution and B) dot-blot of the DB_Interprobe and the product (299-301 bp) of primers DB_16SInterREV and DB_16SInterFWD with genomic DNA of Leptospira fainei serovar Hurstbridge strain BUT6.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/Supp FIGURE_5_EMCC.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 6: (A) Membrane distribution and (B) dot-blot of the DB_16SPathoprobe and the product (1059 bp) of primers DB_16SPathoREV and DB_16SPathoFWD with genomic DNA mix of pathogenic Leptospira serovars.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/Supp FIGURE_6_EMCC.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 7: (A) Membrane distribution and (B) dot-blot of the DB_lipL41probe with the product (479 bp) of primers DB_lipL41REV and DB_lipL41FWD with genomic DNA mix of pathogenic Leptospira serovars.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/Supp FIGURE_7_EMCC.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Alignment of saprophyte Leptospira species with the primers and probe based on the 16S ribosomal RNA sequence. Primers are highlighted in bold and black for their identification, with their direction indicated by black arrows. The probe is indicated in bold and highlighted in grey, and its position is marked with a grey line. The aligned sequences are highlighted in light grey, while the non-corresponding sequences are in black and white.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/SUPPLEMENTARY_FILE_1_EMCC.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Alignment of intermediate Leptospira species with the primers and probe based on the 16S ribosomal RNA sequence. Primers are highlighted in bold and black for their identification, with their direction indicated by black arrows. The probe is indicated in bold and highlighted in grey, and its position is marked with a grey line. The aligned sequences are in light grey, while the non-corresponding sequences are shown in black and white.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/SUPPLEMENTARY_FILE_2_EMCC.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 3: Alignment of pathogenic Leptospira species with the primers and probe based on the lipL32 gene sequence. Primers are highlighted in bold and black for their identification, with their direction indicated by black arrows. The probe is indicated in bold and highlighted in grey, and its position is marked with a grey line. The aligned sequences are in light grey, while the non-corresponding sequences are shown in black and white.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/SUPPLEMENTARY_FILE_3_6th_PR_EMCC.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 4: Alignment of pathogenic Leptospira species with the primers and probe based on the lipL41 gene sequence. For their identification, primers are in bold and highlighted in black, with their direction indicated by black arrows. The probe is indicated in bold and highlighted in dark grey, and its position is marked with a grey line. The aligned sequences are in light grey, while the non-corresponding sequences are in black and white.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/SUPPLEMENTARY_FILE_4_EMCC_V4.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 5: Alignment of Leptospira species with the primers targeting the 16S ribosomal RNA sequence and the probes for saprophyte, intermediate, and pathogenic species. For their identification, primers are indicated in bold and highlighted in black, with their direction marked by black arrows. The probes are indicated in bold and highlighted in grey, with their positions represented by grey lines. The aligned sequences are in light gray, and the non-corresponding sequences are in white and black.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/SUPPLEMENTARY_FILE_5_EMCC_V4.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 6: Buffers and solutions for the dot-blot assay.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65435/SUPPLEMENTARY_FILE_6_8th_PR_EMCC.zip" target="_blank">Please click here to download this File.</a>

Author Spotlight: Leptospira DNA Detection in Water for Environmental Analysis and Disease Surveillance

In this study, a dot-blot application was designed to detect Leptospira from the three main clades in water samples. This method allows for the identification of minimal DNA quantities specifically targeted by a digoxigenin-labeled probe, easily detected by an anti-digoxigenin antibody. This approach is a valuable and satisfactory tool for screening purposes.

Polymerase Chain Reaction and Dot-Blot Hybridization for Leptospira Detection in Water Samples

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by ...

polymerase-chain-reaction-dot-blot-hybridization-for-leptospira

Nanyang Technological University

Research

JoVE Journal

Biology

1.1K Views.  General Hospital “Dr. Manuel Gea González”. Dit protocol is ontworpen om de aanwezigheid van Leptospira-DNA in watermonsters te identificeren. Hiervoor wordt de dot-blot-techniek gebruikt, die een nauwkeurige detectie en analyse van het DNA mogelijk maakt. De dot-blot is een eenvoudige en veelzijdige niet-isotopische techniek en kan helpen bij het beantwoorden van belangrijke vragen over de verspreiding van Leptospira in het milieu.Deze methode heeft het potentieel om enorme waterlichamen te beoordelen en te helpen bij het toezi...

Video: Auteur in de schijnwerpers: Leptospira DNA-detectie in water voor milieuanalyse en ziektebewaking

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. 
Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic &beta;-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.
Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.
The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended 10.

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

Immunology and Infection

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biochemistry

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by pathogenic and virulent bacteria of the genus Leptospira, either by direct contact with the bacteria or indirectly by exposure to contaminated water or soil. Domestic and wild animals act as reservoir hosts of infection, shedding leptospires from colonized renal tubules of the kidney, via urine, into the environment. The generation of mutant strains of Leptospira is critical to evaluate and understand pathogenic mechanisms of infection. CRISPR interference (CRISPRi) has proven to be a straightforward, affordable, and specific tool for gene silencing in pathogenic Leptospira. Therefore, the methodological details of obtaining the plasmid constructs containing both dCas9 and guide RNA, delivery of plasmids to Leptospira by conjugation with the E. coli strain &#946;2163, and transconjugant recovery and evaluation, will be described. In addition, the recently described Hornsby-Alt-Nally (HAN) media allows for the relatively rapid isolation and selection of mutant colonies on agar plates.

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira

Here, the application of CRISPR interference (CRISPRi) for specific gene silencing in Leptospira species is described. Leptospira cells are transformed by conjugation with plasmids expressing dCas9 (catalytically "dead" Cas9) and a single-guide RNA (sgRNA), responsible for base pairing to the desired genomic target. Methods to validate gene silencing are presented.

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by ...

Genetics

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks 

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to humans through the bite of infected Ixodes ticks. The primary etiological agent in North America is Borrelia burgdorferi sensu stricto. As geographic risk regions expand, it is prudent to support robust surveillance programs that can measure tick infection rates, and communicate findings to clinicians, veterinarians, and the general public. The molecular technique of nested polymerase chain reaction (nPCR) has long been used for this purpose, and it remains a central, inexpensive, and robust approach in the detection of Borrelia in both ticks and wildlife. 
This article demonstrates the application of nPCR to tick DNA extracts to identify infected specimens. Two independent B. burgdorferi targets, genes encoding Flagellin B (FlaB) and Outer surface protein A (OspA), have been used extensively with this technique. The protocol involves tick collection, DNA extraction, and then an initial round of PCR to detect each of the two Borrelia-specific loci. Subsequent polymerase chain reaction (PCR) uses the product of the first reaction as a new template to generate smaller, internal amplification fragments. The nested approach improves upon both the specificity and sensitivity of conventional PCR. A tick is considered positive for the pathogen when inner amplicons from both Borrelia genes can be detected by agarose gel electrophoresis.

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Nested PCR is a sensitive, specific, and straightforward technique that can be applied to tick DNA extracts to probe for Borrelia burgdorferi, the causative agent of Lyme disease. The initial PCR experiment uses gene-specific primers to generate long amplicons, which then become templates for a subsequent reaction using internal primers.

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira

For conjugation, Leptospira cells are grown at 29 or 37 degrees Celsius in HAN media. One day before conjugation, donor E. coli cells are grown in LB media supplemented with Diaminopimelic acid - DAP, and spectinomycin.
Next, in the day of conjugation, grow the saturated E. coli cultures in LB plus DAP. Inside a biosafety hood, assemble the filtration apparatus by placing the membrane filter on top of the glass base, followed by 15-mL glass funnel. Both pieces are held together by the spring clamp. Then, connect the glass to a vacuum pump. Add 5 ml of Leptospira cultures to the funnel.
Next, add the E. coli &#946;2163 strain containing the plasmid of interest. The volume will vary according to the optical density of the culture. We are aiming to 1:1 cell proportion. Turn the vacuum pump. Now, liquids will get through the membrane, and cells will be concentrated on top of it.
Take the filter, and place it onto EMJH plate plus DAP, bacterial side up. Incubate the plates at 29 degrees Celsius for 24 hours. Then, recover the filters from the plates, and place it into 50-mL conical tube.
Use 1 mL of liquid HAN media to recover the cells from the filter by pipetting. Visualize the recovered cells by dark-field microscopy.
At this stage, equivalent numbers of E. coli and Leptospires can be seen. 100 to 200 microliters are used to spread the bacteria on two HAN plates containing spectinomycin. At this stage, DAP is omitted, and E. coli will not grow. Plates are incubated at 37 degrees Celsius and 3% CO2. Colonies should be apparent between 8 to 10 days.

Polymerase Chain Reaction and Dot-Blot Hybridization for Leptospira Detection in Water Samples

Samenvatting

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