The authors are grateful to Annette Grendelmeier, Aurelia Drenovac, Cornelia Studer, Daniel Frei, Elisabeth Razavi, Markus Oggenfuss, Seraina Vonzun, and Sven Moeller for participating in the validation of the B. tabaci LAMP assay.

1. Preparations
<ol>
	<li>Preparing aliquots of alkaline DNA extraction solution.
	<ol>
		<li>Produce a stock of alkaline DNA extraction solution using molecular grade water supplemented with 600 &#181;M potassium hydroxide (KOH) and 2 &#181;M Cresol Red. 
		CAUTION: KOH is a strong base dissolved in water. Avoid spills, and skin and eye contact.</li>
		<li>Dispense 30 &#181;L of alkaline DNA extraction solution (prepared in step 1.1.1) into 0.5 mL microcentrifuge tubes and store the aliquots at 4 &#176;C. 
		NOTE: Use the aliquoted DNA extraction solution within 1 year.</li>
	</ol>
	</li>
	<li>Preparing B. tabaci positive amplification control (PAC).
	<ol>
		<li>Generate PCR amplicons of the LAMP target DNA fragment. 
		NOTE: An introduction into general PCR principles and practices is given by Lorenz23.
		<ol>
			<li>Synthesize or obtain the primers C1-J-2195 (5&#8217;-TTGATTTTTTGGTCATCCAGAAGT-3&#8217;) and TL2-N-3014 (5&#8217;-TCCAATGCACTAATCTGCCATATTA-3&#8242;) amplifying a fragment of the mitochondrial COI gene24,25.</li>
			<li>Set up the PCR reaction as described in Table 1. Use DNA extract (see step 2.1) of a reference B. tabaci specimen as DNA template. 
			NOTE: Optionally, it is possible to extract the B. tabaci DNA for the PAC using a commercial kit according to the manufacturer&#8217;s instructions.</li>
			<li>Program a thermal cycler using the following conditions: 15 min at 95 &#176;C; 45 cycles of 40 s at 95 &#176;C, 15 s at 45 &#176;C, ramping over 60 s to 60 &#176;C, 2 min at 72 &#176;C; 7 min at 72 &#176;C; hold at 4 &#176;C.</li>
			<li>Clean the PCR amplification product using a commercial PCR clean-up kit according to the manufacturer&#8217;s protocol and elute the final product in molecular grade water.</li>
			<li>Use a commercial kit with DNA-intercalating dye to measure the DNA concentration of the PCR amplification product according to the manufacturer&#8217;s instructions and dilute with molecular grade water to a concentration of 1 ng/&#181;L. Store the diluted PCR amplification product as PAC stock solution at -20 &#176;C. 
			NOTE: Use the PAC stock solution within 1 year.</li>
			<li>Supplement the PAC stock solution (prepared in step 1.2.1.5) with 0.6 &#181;M KOH and dilute with molecular grade water to a concentration of 5 x 10-3 ng/&#181;L. Store the product at 4 &#176;C. 
			NOTE: Use the PAC within 5 h for the preparation of the ready-to-use B. tabaci LAMP kits described in the next step.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparing ready-to-use B. tabaci LAMP kit (protocol for 20 units)
	<ol>
		<li>Use scissors to cut 8-tube LAMP strips into two 4-tube LAMP strips.</li>
		<li>Label the tubes of the 4-tube LAMP strips according to the scheme shown in Figure 1.</li>
		<li>Prepare B. tabaci LAMP reaction mastermix (protocol for 80 reactions).
		<ol>
			<li>Add 1195.1 &#181;L of ready-to-use GspSSD isothermal master mix (containing GspSSD polymerase, pyrophosphatase, magnesium sulfate, deoxynucleotides, double strand binding DNA binding dye) and 717.4 &#181;L of B. tabaci LAMP primer mix to a 2 mL microcentrifuge tube. Briefly vortex and pulse centrifuge.</li>
			<li>Dispense 22.5 &#181;L of B. tabaci LAMP reaction mastermix (prepared in step 1.3.3.1) into each tube of the 4-tube LAMP strips (prepared in step 1.3.1) and pulse centrifuge.</li>
		</ol>
		</li>
		<li>Vortex the B. tabaci LAMP PAC (prepared in step 1.2) quickly and pulse centrifuge.&#160;Then, add 2.5 &#181;L&#160;into the tube labelled with &#8220;PAC&#8221; of each 4-tube LAMP strip (Figure 1).</li>
		<li>Close lids and store the ready-to-use B. tabaci LAMP kit units at -20 &#176;C. 
		NOTE: Use them within 1 year.</li>
	</ol>
	</li>
</ol>
2. On-site LAMP Analysis
<ol>
	<li>DNA extraction
	<ol>
		<li>Use sterile toothpicks to transfer the insect specimens into 0.5 mL microcentrifuge tubes containing 30 &#181;L of DNA extraction solution (prepared in step 1.1.2). 
		NOTE: Make sure that the insects are immersed in the extraction solution.</li>
		<li>Incubate the samples for 5 min at 95 &#176;C in a thermomixer (300 rpm). Briefly vortex and pulse centrifuge.</li>
	</ol>
	</li>
	<li>B. tabaci LAMP assay
	<ol>
		<li>Thaw a ready-to-use B. tabaci LAMP kit prepared in step 1.3. Vortex quickly and pulse centrifuge. 
		NOTE: With each kit, it is either possible to test two different specimens or to analyze the DNA extract of one specimen in duplicate.</li>
		<li>Add 2.5 &#181;L of sample DNA extract (prepared in step 2.1) into the tubes labeled &#8220;S1&#8221; and &#8220;S2&#8221; of the ready-to-use B. tabaci LAMP kit (Figure 1).</li>
		<li>Add 2.5 &#181;L of pure alkaline DNA extraction solution (prepared in section 1.1) into the tube labeled &#8220;NAC&#8221; for the negative amplification control (Figure 1).</li>
		<li>Vortex the ready-to-use B. tabaci LAMP kit quickly and pulse centrifuge.</li>
		<li>Insert the ready-to-use B. tabaci LAMP kits into the LAMP analysis device (with real-time fluorescence measurement) or a real-time PCR platform and perform an isothermal DNA amplification analysis at 65 &#176;C for 60 min.</li>
		<li>Measure the melting temperatures of DNA amplification products by heating up to 98 &#176;C with a subsequent cooling step (ramp rate of 0.05 &#176;C/s) to 75 &#176;C, while measuring fluorescence in real-time.</li>
	</ol>
	</li>
	<li>LAMP assay read-out
	<ol>
		<li>Validate the LAMP read-out manually as follows.
		<ol>
			<li>If DNA amplifications were measured for the sample and the PAC, no DNA amplification was measured for the NAC, and the annealing temperature of the amplification products were between 80.0 and 85.5 &#176;C, consider the LAMP results as POSITIVE (Figure 2).</li>
			<li>If there is no DNA amplification for the samples (i.e., tubes labeled S1 and S2) but for PAC and NAC then consider the LAMP result as NEGATIVE (Figure 2).</li>
			<li>If DNA amplification was measured for the samples, but the annealing temperatures of corresponding amplification products were outside the range 80.0 &#8210; 85.5 &#176;C, and/or PAC gave no DNA amplification, and/or NAC gave a DNA amplification, consider the LAMP result as INVALID (Figure 2).</li>
		</ol>
		</li>
		<li>Optionally, validate the LAMP read-out using the LAMP validation application (Supplemental file 1).
		<ol>
			<li>Define target species and define the number of tested samples. Click the &#34;Generate Report&#34; button.</li>
			<li>Transfer the read-out (DNA amplification yes/no, annealing temperature amplification product, results of PAC and NAC) from the on-site LAMP analysis device or real-time PCR platform to the corresponding input fields of the validation application. The result of the validation is immediately displayed after entering the data.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>De Barro, P. J., Liu, S. S., Boykin, L. M., Dinsdale, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bemisia+tabaci:+a+statement+of+species+status.">Bemisia tabaci: a statement of species status.</a> Annual Review of Entomology. 56, 1-19 (2011).</li><li>Zhang, G. F., L&#252;, Z. C., Wan, F. H., L&#246;vei, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+quantification+of+Bemisia+tabaci+(Homoptera:+Aleyrodidae)+B-biotype+remains+in+predator+guts.">Real-time PCR quantification of Bemisia tabaci (Homoptera: Aleyrodidae) B-biotype remains in predator guts.</a> Molecular Ecology Notes. 7 (6), 947-954 (2007).</li><li>Boykin, L. M., De Barro, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+identifying+members+of+the+Bemisia+tabaci+species+complex:+and+other+morphologically+identical+species.">A practical guide to identifying members of the Bemisia tabaci species complex: and other morphologically identical species.</a> Frontiers in Ecology and Evolution. 2, (2014).</li><li>Cuthbertson, A. G. S., V&#228;nninen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+maintaining+protected+zone+status+against+Bemisia+tabaci.">The importance of maintaining protected zone status against Bemisia tabaci.</a> Insects. 6 (2), 432-441 (2015).</li><li>Dalmon, A., Halkett, F., Granier, M., Delatte, H., Peterschmitt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+structure+of+the+invasive+pest+Bemisia+tabaci:+evidence+of+limited+but+persistent+genetic+differentiation+in+glasshouse+populations.">Genetic structure of the invasive pest Bemisia tabaci: evidence of limited but persistent genetic differentiation in glasshouse populations.</a> Heredity. 100 (3), 316-325 (2008).</li><li>Dickey, A. M., Osborne, L. S., Shatters, R. G., Hall, P. A. M., Mckenzie, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+genetics+of+invasive+Bemisia+tabaci+(Hemiptera:+Aleyrodidae)+cryptic+species+in+the+United+States+based+on+microsatellite+markers.">Population genetics of invasive Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species in the United States based on microsatellite markers.</a> Journal of Economic Entomology. 106 (3), 1355-1364 (2013).</li><li>Bacon, S. J., Bacher, S., Aebi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaps+in+border+controls+are+related+to+quarantine+alien+insect+invasions+in+Europe.">Gaps in border controls are related to quarantine alien insect invasions in Europe.</a> PLOS ONE. 7 (10), e47689 (2012).</li><li>Blaser, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+laboratory+to+point+of+entry:+development+and+implementation+of+a+loop-mediated+isothermal+amplification+(LAMP)-based+genetic+identification+system+to+prevent+introduction+of+quarantine+insect+species.">From laboratory to point of entry: development and implementation of a loop-mediated isothermal amplification (LAMP)-based genetic identification system to prevent introduction of quarantine insect species.</a> Pest Management Science. 74 (6), 1504-1512 (2018).</li><li>Floyd, R., Lima, J., de Waard, J., Humble, L., Hanner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+goals:+policy+implications+of+DNA+barcoding+as+a+protocol+for+identification+of+arthropod+pests.">Common goals: policy implications of DNA barcoding as a protocol for identification of arthropod pests.</a> Biological Invasions. 12 (9), 2947-2954 (2010).</li><li>Armstrong, K. F., Ball, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+barcodes+for+biosecurity:+invasive+species+identification.">DNA barcodes for biosecurity: invasive species identification.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 360 (1462), 1813-1823 (2005).</li><li>Notomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Research. 28 (12), E63 (2000).</li><li>Tomlinson, J. A., Boonham, N., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+one-hour+DNA+extraction+and+loop-mediated+isothermal+amplification+assay+for+rapid+detection+of+phytoplasmas.">Development and evaluation of a one-hour DNA extraction and loop-mediated isothermal amplification assay for rapid detection of phytoplasmas.</a> Plant Pathology. 59 (3), 465-471 (2010).</li><li>Kogov&#353;ek, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LAMP+assay+and+rapid+sample+preparation+method+for+on-site+detection+of+flavescence+dor&#233;e+phytoplasma+in+grapevine.">LAMP assay and rapid sample preparation method for on-site detection of flavescence dor&#233;e phytoplasma in grapevine.</a> Plant Pathology. 64 (2), 286-296 (2015).</li><li>Lee, M. S., Su, T. Y., Lien, Y. Y., Sheu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+loop-mediated+isothermal+amplification+(LAMP)+assays+for+the+rapid+authentication+of+five+forbidden+vegetables+in+strict+vegetarian+diets.">The development of loop-mediated isothermal amplification (LAMP) assays for the rapid authentication of five forbidden vegetables in strict vegetarian diets.</a> Scientific Reports. 7, 44238 (2017).</li><li>B&#252;hlmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erwinia+amylovora+loop-mediated+isothermal+amplification+(LAMP)+assay+for+rapid+pathogen+detection+and+on-site+diagnosis+of+fire+blight.">Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight.</a> Journal of Microbiological Methods. 92 (3), 332-339 (2013).</li><li>Cavalieri, V., Manglli, A., Tiberini, A., Tomassoli, L., Rapisarda, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+Trialeurodes+vaporariorum,+Bemisia+tabaci+(MEAM1+and+MED)+and+tomato-infecting+criniviruses+in+whiteflies+and+in+tomato+leaves+by+real-time+reverse+transcription-PCR+assay.">Rapid identification of Trialeurodes vaporariorum, Bemisia tabaci (MEAM1 and MED) and tomato-infecting criniviruses in whiteflies and in tomato leaves by real-time reverse transcription-PCR assay.</a> Bulletin of Insectology. 67 (2), 219-225 (2014).</li><li>Baek, J. H., Lee, H. J., Kim, Y. H., Lim, K. J., Lee, S. H., Kim, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+antibody-based+diagnostic+method+for+the+identification+of+Bemisia+tabaci+biotype+B.">Development of an antibody-based diagnostic method for the identification of Bemisia tabaci biotype B.</a> Pesticide Biochemistry and Physiology. 131, 18-23 (2016).</li><li>Hsieh, C. H., Wang, H. Y., Chen, Y. F., Ko, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+rapid+identification+of+biotypes+B+and+Q+of+the+globally+invasive+pest+Bemisia+tabaci,+and+studying+population+dynamics.">Loop-mediated isothermal amplification for rapid identification of biotypes B and Q of the globally invasive pest Bemisia tabaci, and studying population dynamics.</a> Pest Management Science. 68 (8), 1206-1213 (2012).</li><li>Rejili, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PCR-based+diagnostic+assay+for+detecting+DNA+of+the+olive+fruit+fly,+Bactrocera+oleae,+in+the+gut+of+soil-living+arthropods.">A PCR-based diagnostic assay for detecting DNA of the olive fruit fly, Bactrocera oleae, in the gut of soil-living arthropods.</a> Bulletin of Entomological Research. 106 (5), 695-699 (2016).</li><li>Hebert, P. D. N., Ratnasingham, S., deWaard, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barcoding+animal+life:+cytochrome+c+oxidase+subunit+1+divergences+among+closely+related+species.">Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species.</a> Proceedings. Biological Sciences. 270 (Suppl 1), 96-99 (2003).</li><li>Ratnasingham, S., Hebert, P. D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bold:+The+Barcode+of+Life+Data+System+(http://www.barcodinglife.org).">bold: The Barcode of Life Data System (http://www.barcodinglife.org).</a> Molecular Ecology Notes. 7 (3), 355-364 (2007).</li><li>Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., Bank Wheeler, D. L. G. e. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GenBank.">GenBank.</a> Nucleic Acids Research. 33, D34-D38 (2005).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> Journal of Visualized Experiments. (63), e3998 (2012).</li><li>Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution,+weighting,+and+phylogenetic+utility+of+mitochondrial+gene+sequences+and+a+compilation+of+conserved+polymerase+chain+reaction+primers.">Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers.</a> Annals of the Entomological Society of America. 87 (6), 651-701 (1994).</li><li>Simon, C., Buckley, T. R., Frati, F., Stewart, J. B., Beckenbach, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporating+molecular+evolution+into+phylogenetic+analysis,+and+a+new+compilation+of+conserved+polymerase+chain+reaction+primers+for+animal+mitochondrial+DNA.">Incorporating molecular evolution into phylogenetic analysis, and a new compilation of conserved polymerase chain reaction primers for animal mitochondrial DNA.</a> Annual Review of Ecology, Evolution, and Systematics. 37 (1), 545-579 (2006).</li><li>Harper, S. J., Ward, L. I., Clover, G. R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+LAMP+and+real-time+PCR+methods+for+the+rapid+detection+of+Xylella+fastidiosa+for+quarantine+and+field+applications.">Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications.</a> Phytopathology. 100 (12), 1282-1288 (2010).</li><li>Lauri, A., Mariani, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentials+and+limitations+of+molecular+diagnostic+methods+in+food+safety.">Potentials and limitations of molecular diagnostic methods in food safety.</a> Genes &amp; Nutrition. 4 (1), 1-12 (2009).</li><li>Tomlinson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+loop-mediated+isothermal+amplification-based+method+for+confirmation+of+Guignardia+citricarpa+in+citrus+black+spot+lesions.">A loop-mediated isothermal amplification-based method for confirmation of Guignardia citricarpa in citrus black spot lesions.</a> European Journal of Plant Pathology. 136 (2), 217-224 (2013).</li><li>Branton, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+and+challenges+of+nanopore+sequencing.">The potential and challenges of nanopore sequencing.</a> Nature Biotechnology. 26 (10), 1146-1153 (2008).</li><li>Pomerantz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+DNA+barcoding+in+a+rainforest+using+nanopore+sequencing:+opportunities+for+rapid+biodiversity+assessments+and+local+capacity+building.">Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building.</a> GigaScience. 7 (4), giy033 (2018).</li></ol>

The author Michael Andreou is a shareholder of OptiGene Limited that produces reagents and instruments used in this article. The other authors have nothing to disclose.

The ability to accurately identify potentially harmful organisms without time delay represents a critical aspect for the management of pest species9,10,26. Besides being rapid, for plant import products, an ideal pest identification method should be simple to perform on-site at POEs8,26. This paper reports the protocol of a novel LAMP assay for the rapid identification of B. tabaci, a quarantine insect organism frequently intercepted at European borders (https://ec.europa.eu/food/sites/food/files/plant/docs/ph_biosec_europhyt_annual-report _2016.pdf).
The rationale behind the development of the diagnostic test was to design an easy-to-follow protocol which can be performed during the plant import control procedure by plant health inspectors with minimal laboratory training. In order to make on-site testing as rapid and simple as possible, the protocol is divided into two parts, the preparation of a ready-to-use kit and the actual performance of the LAMP assay. The first part may be done in an external laboratory so that the plant health inspector can perform the DNA extraction and LAMP assay on-site with only one pipetting step.
Though only one step, pipetting small amounts of liquid may be challenging for users with little or no laboratory experience. To address this issue, a dye (cresol red) is added to the extraction solution so that the operator can visually confirm the small amount (i.e., 2.5 &#181;L) of DNA is correctly transferred to the respective tube. Another important simplification of the protocol is the validation application as it facilitates a reliable interpretation of the LAMP read-out (Supplemental file 1).
The novel B. tabaci LAMP assay has been validated under laboratory and on-site conditions by testing insect specimens intercepted during the regular import control process of Switzerland8. In total, 80 specimens from three continents, Africa, Eurasia, and North America, were analyzed by LAMP. Of the 80 specimens, only three (3.8%) were wrongly identified (false-negatives)8. When analyzing the primer target DNA sequences of the false-negative specimens, it was found that they were new B. tabaci haplotypes that have so far not been described8. Based on these results, the B. tabaci LAMP primer set has been modified and successfully re-validated8.
One major limitation of any DNA amplification-based method including LAMP is that they only identify pre-defined target DNA sequences8,27. A comprehensive knowledge of the genetic variation found in the primer target sequence is therefore crucial to ensure diagnostic accuracy8,27. However, such information is often very limited, especially in the case of newly emerging pest species8. Though rare, false-negative results caused by mutations in the target sequence are expected8. In the case of the present B. tabaci LAMP assay, a solution for this problem is the combination with a DNA barcoding-based technology, a strategy realized in the course of the implementation of this diagnostic test at the POE Zurich Airport8. Here, all LAMP-negative results were re-analyzed by DNA barcoding in an external laboratory8. In case a novel pest haplotype not yet described is encountered, the LAMP primers can be modified using the DNA sequence generated in the barcoding process8. Thereby, the resulting loss of speed in case of a negative LAMP result is compensated for the maximum diagnostic accuracy ensured in this two-stage process8.
The set-up costs for the current LAMP assay at a POE are approximately USD 25,000. With the increasing number of LAMP tests developed for plant pests (e.g., Erwinia amylovora, Flavescence dor&#233;e, and&#160;Guignardia citricarpa), such a one-time investment appears justified13,15,28. However, the protocol could potentially be modified to reduce these costs even further. For example, for the DNA extraction step at 95 &#176;C the thermo mixer used here could be replaced by a less expensive water bath, or by performing this step directly in the real time LAMP device. Furthermore, the mixing steps on the vortex could probably be replaced by manually flicking the tubes, and in the DNA transfer step the pipettor might be replaced by sterile inoculation loops.
Future improvements for a rapid identification of B. tabaci and pest species in general could be an implementation of an on-site sequencing approach that would allow to perform DNA barcoding analyses at POEs. A promising candidate system for such an implementation is the nanopore sequencing technology. Indeed, the technology has recently been successfully implemented in an on-site DNA barcoding effort to assess the biodiversity of a rainforest8,29,30. An on-site DNA barcoding identification system can completely replace the need for the development of targeted diagnostic tests and their validation. Also it allows collecting additional information about pest characteristics such as pesticide resistance genes8. Nevertheless, until novel sequencing technologies will be implemented routinely, the B. tabaci LAMP assay represents a rapid (&#60;1 h) and accurate identification method.

The whitefly Bemisia tabaci (Gennadius) is an invasive insect pest affecting the yield of many economically important crops including ornamental plants, vegetables, grain legumes, and cotton1,2. Beside damage caused through direct phloem-feeding, the homopteran species harms plants indirectly by the excretion of large amounts of honeydew onto the surfaces of leaves and fruits, as well as by the transmission of numerous plant pathogenic viruses1,3,4. Recent genetic studies comparing DNA sequences of the mitochondrial gene cytochrome c oxidase 1 (COI) revealed that B. tabaci is a species complex of at least 34 morphocryptic species3,4. Two highly invasive and damaging members within this complex, biotype B originating from the Middle East and the Asian Minor region, as well as biotype Q originating from the Mediterranean region, have been dispersed globally through international trading activities with plant products, particularly by the transportation of ornamentals1,5,6. Due to its worldwide pest status, the International Union for the Conservation of Nature and Natural Resources (IUCN) listed B. tabaci as one of the &#34;world&#39;s 100 worst invasive alien species&#34; and members of the species complex are regulated organisms by many countries1,3,4.
In the European Union (EU), B. tabaci is listed in the Plant Health Directive 2000/29/EC Annex 1AI as a quarantine organism whose introduction from non-EU countries and its dissemination within the EU are banned4. An essential prevention measure against the spread of quarantine organisms is the inspection of plant shipments at points of entry (POEs) such as airports and seaports7,8. In the case a quarantine organism is found, the National Plant Protection Organization (NPPO) in charge takes action by either rejecting or treatment (including destruction) of the infested shipment9. However, officers inspecting the imports often do not have the taxonomic expertise to accurately identify the vast range of pest species associated with global trade9. Especially the identification of immature life stages (e.g., eggs and larvae) without distinct morphological keys is virtually impossible for non-taxonomists8,9,10. Consequently, to enable implementation of quarantine measures with minimal delay, there is a need for alternative, rapid on-site identification assays9.
A candidate method is the loop-mediated isothermal DNA amplification (LAMP) technology that has recently been shown to be a suitable technology for the identification of plant pathogens11,12,13. LAMP is highly specific because the method uses at least two primer pairs recognizing six distinct DNA target sequences14. Due to the DNA strand displacement activity of the Bst DNA polymerase, LAMP reactions are performed under isothermal conditions14. Hence, in contrast to conventional polymerase chain reaction (PCR)-based assays there is no need for a thermal cycler13,14. Another advantage over PCR-based assays is its resilience against potential inhibitors in the DNA extract, circumventing the need for a DNA purification step13. Due to the protocol&#39;s speed and simplicity, LAMP may even be performed under on-site conditions using a portable, battery driven real-time detection device8,15.
A LAMP assay was designed in response to the demand for a rapid on-site identification method for B. tabaci8. The overarching aim was to develop a protocol that can be performed by plant health inspectors with limited laboratory training. A strong focus was, therefore, set on optimizing speed and simplicity of the protocol. While existing diagnostic tests have generally been developed for the identification of one or several biotypes of B. tabaci, the novel LAMP assay covers the whole B. tabaci species complex8,16,17,18. The problem of the pronounced genetic within-taxon diversity of the complex was solved by using combinations of different primer sets and the application of degenerate primers8. The novel B. tabaci LAMP assay is designed in such a way that the primers target a fragment at the 3&#39; end of the mitochondrial COI gene8. This gene presents a suitable target for animal diagnostic assays because it harbors regions conserved enough to ensure diagnostic sensitivity for a specific species, while discriminating enough between closely related organisms19,20. Furthermore, the COI gene is often used as a genetic marker in population genetic studies and as a signature sequence in DNA barcoding analyses, resulting in numerous DNA sequence entries in open source databases such as GenBank and BOLD21,22. Beside the publicly available COI sequences from B. tabaci, COI sequences from closely related species (Aleurocanthus spp. [N = 2], Aleurochiton aceris, Aleurodicus dugesii, Bemisia spp. [N = 3], Neomaskellia andropogonis, Tetraleurodes acaciae, and Trialeurodes spp. [N = 4]) were included in the primer design of this study and used to assess diagnostic sensitivity and specificity in silico8.
Due to the accuracy of the method, its speed (&#60;1 h) and the simplicity of the protocol, the assay has been shown to be suitable for on-site application when implemented as part of the import control procedure at a Swiss POE8.

<table><tbody><tr><td>&lt;em&gt;B. tabaci&lt;/em&gt; LAMP primer mix</td><td>OptiGene Ltd.</td><td>on request</td><td>For preparation of&lt;br/&gt; read-to-use &lt;em&gt;B. tabaci &lt;/em&gt;LAMP kit</td></tr><tr><td>Centrifuge MiniSpin</td><td>Eppendorf AG</td><td>5452000010</td><td>For several centrifugation steps</td></tr><tr><td>Cresol red (red dye)</td><td>Sigma-Aldrich Corp.</td><td>114472</td><td>Component of&lt;br/&gt; DNA extraction solution</td></tr><tr><td>Eppendorf ThermoMixer</td><td>Eppendorf AG</td><td>5382000015</td><td>For DNA extraction</td></tr><tr><td>Genie II (on-site LAMP analysis device)</td><td>OptiGene Ltd.</td><td>Genie&amp;reg; II</td><td>For LAMP analysis</td></tr><tr><td>Genie Strips (8-tube LAMP strips)</td><td>OptiGene Ltd.</td><td>OP-0008-50</td><td>For preparation of&lt;br/&gt; read-to-use &lt;em&gt;B. tabaci &lt;/em&gt;LAMP kit</td></tr><tr><td>HotStarTaq Master Mix</td><td>Qiagen AG</td><td>203443</td><td>For generation of positive&lt;br/&gt; amplification control</td></tr><tr><td>Labcycler (Thermocycler)</td><td>SensoQuest GmbH,&lt;br/&gt; distributed by Witec AG</td><td>011-103</td><td>For DNA extraction</td></tr><tr><td>GspSSD Lyse n&#039; Lamp Isothermal Mastermix</td><td>OptiGene Ltd.</td><td>ISO-001LNL</td><td>For preparation of&lt;br/&gt; read-to-use &lt;em&gt;B. tabaci &lt;/em&gt;LAMP kit</td></tr><tr><td>Mini centrifuge Labnet Prism</td><td>Labnet International Inc.</td><td>C1801</td><td>For several centrifugation steps</td></tr><tr><td>NucleoFast 96 PCR</td><td>Marcherey-Nagel GmbH</td><td>743500.4</td><td>For clean-up of positive&lt;br/&gt; amplification control</td></tr><tr><td>Potassium hydroxide solution</td><td>Sigma-Aldrich Corp.</td><td>319376</td><td>Component of&lt;br/&gt; DNA extraction solution</td></tr><tr><td>Qbit Fluorometer 3</td><td>Thermo Fisher Scientific Corp.</td><td>Q33226</td><td>For measuring DNA conentration&lt;br/&gt; of positive control</td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher Scientific</td><td>Q32854</td><td>For clean-up of positive&lt;br/&gt; amplification control</td></tr><tr><td>Safe-Lock Tubes 0.5 mL (microcentrifuge tube)</td><td>Eppendorf AG</td><td>0030 121.023</td><td>For DNA extraction</td></tr><tr><td>Safe-Lock Tubes 2.0 mL (microcentrifuge tube)</td><td>Eppendorf AG</td><td>0030 120.094</td><td>For preparation of&lt;br/&gt; read-to-use B. tabaci LAMP kit</td></tr><tr><td>Wood Toothpicks</td><td>VWR International LLC</td><td>470226-594</td><td>For DNA extraction</td></tr><tr><td>Vortex-Genie 2 (Vortex)</td><td>Scientific Industries Inc.</td><td>SI-0236</td><td>For several mixing steps</td></tr></tbody></table>

a loop-mediated isothermal amplification (lamp) assay for rapid identification of bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

During the validation of the B. tabaci LAMP assay, insect specimens intercepted in the course of the regular Swiss import control process were analyzed8. The specimens originated from eight different countries (Canary Islands, Dominican Republic, Israel, Malaysia, Morocco, Singapore, Thailand, and Vietnam) and reflect the genetic diversity of B. tabaci found at European POEs8. All LAMP results were cross-validated by DNA barcoding8.
From a total of 80 specimens analyzed by LAMP, 75 specimens (93.8%) were correctly identified as B. tabaci (true-positives), two specimens (2.5%) were correctly identified as not being B. tabaci (true-negatives), and three specimens (3.8%) were wrongly identified as not being B.&#160;tabaci (false-negatives) (Table 2)8. The correct-negative results originated from two Trialeurodes vaporariorum specimens, a non-regulated species at high risk to be confused with B. tabaci at POEs for plant products8. Based on these results, the following measurements of diagnostic accuracy were calculated: test specificity (true-negative rate), 100%; test sensitivity (true-positive rate), 96.2%; test efficiency (percentage of correct test results), 96.3% (Table 2)8. When assessing the analytical sensitivity (detection limit), the B. tabaci LAMP assay successfully amplified sample DNA diluted to 100 fg/&#181;L across three technical replicates (Table 3).
A subset of the assays (N = 13) was performed under on-site conditions at the Swiss POE Zurich Airport by plant health inspectors using the ready-to-use B. tabaci LAMP kits8. When cross-validated in the reference laboratory, all results from on-site testing were found to be correct (test efficiency = 100%)8. Assessing the on-site LAMP assay performance, the average time to positive (time until a positive results was available) was 38.4 &#177; 10.3 min (mean &#177; standard deviation)8. A representative DNA amplification plot and the corresponding annealing derivative from a B. tabaci LAMP analysis performed under on-site conditions are shown in Figure 3A and B. In this example, sample one and two were correctly identified as B. tabaci indicated by DNA amplification after approximately 30 min (Figure 3A) together with the expected annealing temperatures at approximately 82 &#176;C (Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58502/58502fig1.jpg" /> 
Figure 1: Visualization of the experimental set-up of a ready-to-use B. tabaci LAMP kit described in the protocol. S1, sample 1; S2, sample 2; PAC, positive amplification control; NAC, negative amplification control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58502/58502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58502/58502fig2.jpg" /> 
Figure 2: LAMP read-out validation schema. PAC: positive amplification control; NAC: negative amplification control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58502/58502fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58502/58502fig3.jpg" /> 
Figure 3: DNA amplification plot (A) and annealing derivative (B) of a B. tabaci LAMP analysis performed under on-site conditions. Fluorescence was measured in relative intensity units. Green line, sample 1; orange line, sample 2; blue line, negative amplification control (NAC); red line, positive amplification control (PAC). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58502/58502fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Stock conc.</td>
			<td>Final reaction conc.</td>
			<td>Volume per reaction</td>
		</tr>
		<tr>
			<td>Taq Polymerase Master Mix&#160;&#160;</td>
			<td>2x</td>
			<td>1x</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>Primer C1-J-2195</td>
			<td>20 &#181;M</td>
			<td>0.4 &#181;M</td>
			<td>0.4 &#181;L</td>
		</tr>
		<tr>
			<td>Primer TL2-N-3014</td>
			<td>20 &#181;M</td>
			<td>0.4 &#181;M</td>
			<td>0.4 &#181;L</td>
		</tr>
		<tr>
			<td>Molecular Grade Water</td>
			<td>-</td>
			<td>-</td>
			<td>8.2 &#181;L</td>
		</tr>
		<tr>
			<td>DNA Template</td>
			<td>-</td>
			<td>-</td>
			<td>1 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Preparation of PCR reaction mastermix for the B. tabaci positive amplification control. Components and concentrations needed to set up one PCR reaction. The final reaction volume is 20 &#181;L. Primer sequences are shown in 1.2.1.1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>N</td>
			<td>NTP</td>
			<td>NFP</td>
			<td>NTN</td>
			<td>NFN</td>
			<td>SEN (%)</td>
			<td>SPE (%)</td>
			<td>EFF (%)</td>
		</tr>
		<tr>
			<td>80</td>
			<td>75</td>
			<td>0</td>
			<td>2</td>
			<td>3</td>
			<td>96.2</td>
			<td>100</td>
			<td>96.3</td>
		</tr>
	</tbody>
</table>
Table 2: Results of the B. tabaci LAMP assay validation. N, number of analyses; NTP, number of true-positive results; NFP, number of false-positive results; NTN, number of true-negative results; NFN, number of false-negative results; SEN, diagnostic sensitivity; SPE, diagnostic specificity, EFF, test efficiency.&#160;
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CDNA (fg/&#181;L)</td>
			<td>NPR</td>
			<td>TP (min) 
			(mean &#177; SD)</td>
			<td>TA (&#176;C) 
			(mean &#177; SD)</td>
		</tr>
		<tr>
			<td>1 x 105</td>
			<td>3</td>
			<td>33.5 &#177; 2.9</td>
			<td>81.3 &#177; 0.1</td>
		</tr>
		<tr>
			<td>1 x 104</td>
			<td>3</td>
			<td>30.7 &#177; 1.1</td>
			<td>81 &#177; 0.0</td>
		</tr>
		<tr>
			<td>1 x 103</td>
			<td>3</td>
			<td>40.4 &#177; 3.9</td>
			<td>81.1 &#177; 0.1</td>
		</tr>
		<tr>
			<td>1 x 102</td>
			<td>3</td>
			<td>50.7 &#177; 1.6</td>
			<td>81.1 &#177;0.1</td>
		</tr>
		<tr>
			<td>1 x 101</td>
			<td>0</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>1 x 100</td>
			<td>0</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 3: Analytical sensitivity (detection limit) of the B. tabaci LAMP assay. Each dilution was tested in triplicates. CDNA, DNA concentration per reaction; NPR, number of positive replicates; TP, time until a positive result was available; TA, annealing temperature; SD, standard deviation.

Watch this Scientific Journal Video about A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci at JoVE.com

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

a-loop-mediated-isothermal-amplification-lamp-assay-for-rapid

Universidad San Sebastian

Research

JoVE Journal

Environment

16.5K Views.  Agroscope. Deze methode biedt een snel en betrouwbaar diagnostisch instrument voor de identificatie van Bemisia tabaci, een invasieve insectenplaag die de productie van groenten en siergewassen in veel landen over de hele wereld beïnvloedt. Vanwege het gemak kan de methode ter plaatse rechtstreeks worden uitgevoerd op plaatsen van binnenkomst voor plantaardige importproducten, zoals havens en luchthavens, en heeft daardoor het potentieel om de verspreiding van Bemisia tabaci langs handelsnetwerken te v...

Video: A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

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

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Genetics

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Immunology and Infection

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video