HIV drug resistance has the potential to derail the progress made in the efforts to control the HIV epidemic. Therefore, it is important to continuously survey and monitor drug resistance in naive patients and patients failing therapy. Unfortunately, most of the methods currently used expensive to implement, especially in the context of the magnitude of the HIV epidemic in Sub-Saharan Africa.
Here we demonstrate a low cost and open access method for HIV drug resistance genotyping. This protocol is divided into four stages. RNA extraction, the best transcription and PCR sequencing and bioinformatics.
H-I-V-R-N-A is extracted using the KaiGen viral RNA kit. The method uses columns to trap and clean precipitated. RNA at this stage always include positive and negative controls.
Before starting, calculate the volumes of each of the regions required. For the number of samples being processed, it is recommended to always add a region control. In addition to the extracted positive and negative controls, prepare the DNTP primer mix by adding 0.5 microliters of the primer RT 21 and 0.5 microliters of the DNTP mix to a clean sterile 200 microliter PCR tube, followed by briefly.
For taxing, prepare t enzyme mix by adding one microliter of the 10 times buffer, one microliter of 3.1 molar DTT and two microliters of 25 millimolar. Magnesium chloride to sterile tube add 0.5 microliters of each of the enzymes, RNAs out and s script. Three, reverse transcripts to the enzyme mix tube, followed by a gentle mix.
Keep the tubes with a dent p primer mixes and enzyme mix on a code block and move to the RNA station tors of the RNA to the DNTP prima mix tube. After the addition of the RNA, move to the PCR room with all the tubes on a card block and refuge the DNTP primary RNA mix and place them into a thermocycler heat. Exclude five degrees Celsius for five minutes and rapidly cool to four degrees Celsius.
Hold for two minutes. Pause the thermocycler whilst two at four degrees Celsius. Take out the tubes and add five microliters of the enzyme mix while keeping the tubes on a code block mix gently followed by briefly centrifuging and return to the thermocycler.
Hold the tubes at 50 degrees Celsius for 60 minutes, followed by eight five degrees Celsius for five minutes. Cool to 37 degrees Celsius. And pause as soon as it reaches this temperature.
Take the tubes out of the thermocycler quickly. Add open five microliters of RNAs edge to the tubes and return the thermo cycle. Hold 37 degrees Celsius for 20 minutes and then code four degrees Celsius before starting.
Calculate the volumes of each of the regions required with the number of samples being processed and the controls in addition to the three controls, positive, negative, and the reagent. You can also add a PCR control the first and second round. PCR mixes can be prepared simultaneously and the second master mix.
Third, at minus credit degree sources until needed. Mixes can be stored for up to eight hours. Add the water 10 times buffer, magnesium chloride, DTPs and primers in the mouth shown on the table.
And vortex. Add 0.1 microliters of platinum tag polymerase and gently mix early cord 23 microliters of the master mix to 200 microliter PCR tubes with the master mix tubes on a cord block. Move to the PCR room.
Add two microliters of the CDNA to 23 microliters of the first round PCR master mix. Close the tubes, put the samples in the thermocycler and run the PCR program continued. Second round PCR stage or store the first round PCR products at minus 20 degrees Celsius or colder until required.
For second round PCR, add two microliters of the first round PCR product to 23 microliters of the second round PCR master mix and use the same PCR program to evaluate the PCR amplification. Perform 1%a electrophoresis 800 volts and 400 words for 40 minutes. Positive amplification can be visualized as a 1, 315 base per fragment.
There should be no amplification in the negative and reagent controls in preparation for the sequencing reaction. The positive second round PCR products are cleaned up. Using the pure link PCR purification kit, add 80 microliters of binding buffer to 20 microliters of PCR product and pipette mix.
Add the sample mixed with the bending buffer to a pure link spin column in a collection tube. Centrifuge the column at 10, 000 RCF for one minute. Transfer the column into a new collection tube.
Wash the column with 650 microliters of wash buffer centrifuge. The column at 10, 000 r save for one minute. Transfer the column into a new collection tube.
F the column, its maximum speed for two to three minutes. Place a spin column in a clean 1.7 milliliter elucian tube. Add 40 microliters of elucian buffer to the center of the column and incubate the column at room temperature for one minute.
Centrifuge the column at maximum speed for two to three minutes. The elucian tube contains your purified PCR product ready for sequencing. Discard the column.
The PCR products are sequenced using the big D terminator kit version 3.1 and four primers for each sample For each of the primers. Set up the sequencing reactions as indicated on the table. Gently mixed the master mix by invading the tubes early.
Caught nine microliters of the master mix to a 96 wheel plate. One plate will run 24 samples. You can use layout below in which each column accommodates two samples and one microliter of the DNA sample.
Cover the plate and gently mix centrifuge in 3000 RCF for one minute. Place the plate on the thermocycler and run the cycling program where the PCR finishes. Clean up the sequencing product immediately.
The byproducts of sequencing reactions as well as excess contaminators can interfere with sequencing electrophoresis. It is therefore important to clean up the sequencing products to remove any excess premise and incorporated d terminators salts and enzymes before loading the samples for automated fluorescent DNA sequencing electrophoresis. The method used here is a simple e ethanol sodium ASCE protocol for each sequencing reaction makes 50 microliters of absolute ethanol and five microliters of three molar sodium acid.
Using a multichannel prepared add fit five microliters of sodium acid ethanol solution to each. Well seal wells with adhesive foil ensuring that each well is sewed properly and vortex to mix centrifuge at 3000 RCF for 20 minutes. After 20 minutes, remove cover and in one smooth motion invade the plate onto a forward che wipe.
Fuge the inverted plate at 150 RC for two minutes immediately add 150 microliters of code ethanol to not delay addition of ethanol at this step seal with the same adhesive foil cover and vortex centrifuge at 3000 RCF for five minutes. Invert over folded chemo repair and centrifuge inverted plate at 150 RCF for one minute after the ification place uncovered in a thermocycler and dry it at 50 degree Celsius. Once dry seal the place with adhesive foil covers wrap in foil and store it minus 20 degrees Celsius and until ready to proceed with the sequencing electrophoresis.
When ready to sequence dissolve infor amide denature and load onto the 31 30 genetic analyzer for electrophoresis. Launch the program genius. Create a working folder inside the local documents to store the sequences.
Import the A BF files generated by the sequencing machine to the genius working Folder. Using the import tool genius will allocate percentage quality scores for each sequence Imported open sequences with quality scores of greater than 70%By double clicking on them, each file should open in a new window. Genius will indicate the quality each nucleotide position of the sequence using light blue buzz.
The higher the bar, the better the quality of the best score. Using your er, select the midsection of the sequence leaving out the ends. Click on the extract button to extract the region with high quality sequence.
Select all four extracted sequences for each sample and assemble them against an Annotated reference sequence. Inspect the assembled sequence to Ensure that you are in the correct reading frame. If you are in the correct reading frame, the beginning of subtype c protease should start with the following.Amen.
Acids P-Q-I-L-W-T. The beginning of RT will start with P-I-S-P-I-A extract the CONT region covering the beginning of to the 300th RT cord. During this process, also check for insertions or deletions.
Go through the consensus sequence of the extracted conti, identifying any ambiguities and verify positions with mixed bases by inspecting quality symmetry heights, background and shoulders of the flanking regions of the basis. Select consensus sequence and click the extract button to create a separate file of the consensus sequence generated from the four premise and label it appropriately. Expose the sequence to a backup Storage folder on the computer or network.
Analyze the sequences using the H HIV V DB program@hivdb.stanford.edu. Check for deletions and in Sessions on the ARI data in that. The sequence covers all the 99 protest codes in the phase 300 RT codes.
Check for any highlighted QA issues in both the protests in RT regions, such as top codes, frame shifts, ambiguous positions, and unusual residues, plus the new sequence against a local Sequence database from previous runs. If the new sequence is Greater than 97%similar to any of the sequence in the database, all the stages of the protocol should be reviewed. Starting with sequence analysis, going back to the RNA extraction to ensure that there were no mixed up or contamination.
If no problems are identified, resequence both the old and the new samples if the sequences are still greater than 97%similar. Review the patient history to assess for any epidemiologic linkage between the patients. This is also another quality tool that is employed to identify contamination.
Simple mix up or samples from epidemiological linked patients. Align all the sequences from the database using the Cluster W program. Ingenious manually check the alignment for misaligned sequences, deletions, and in sessions and edit accordingly.
Construct a phylogenetic tree using Genius three builder or other tree builders ingenious. Examine the tree for samples with short branch length. Review the samples with short branch length for Possible contamination.
Log into the REGAR DB using a unique username and password on the Dropdown menu. Under patient ID.Select begins with a the patient ID and select the patient whose sequence is to be applauded on the menu. To right select viral isolate from the options under viral select add.
Enter the sample dates, sample id, sequence ID and sequencing date. Select choose file, and then navigate to the faster file to be applauded. After selecting the faster file to be applauded, click on applaud.
Once the applauded sequence appears in the CLE box, click the okay button at the bottom right of the window. Check for produce in rt. Pot alignments by clicking on protein and selecting either PR or rt.
Check for the drug resistance mutations using the resistance pattern. This gives you the resistance profiles from three algorithms, A NRS, Stanford, HIV DB, and rega db. Click on the viral isolate reports tab.
Select the algorithms for the interpretation of the genotype and the reports templates to use. Once the algorithm and the template are selected, click on generics. Download the RTF document generated.
Open the RTF Document as a word document. Recite The treatment history chart after the chart, A, the section Clinical chart and resistance interpretation. Add a description of the patient's treatment history and the viral resistance profile.
Also, add a description of the patient's viral load and CD four profiles. Send the report to add the specialist for review and recommendations on Future patient management. The reduction In the overall cost of this protocol was achieved through four different strategies.
The first one was killing down the reaction volumes in almost every stage, thereby allowing more efficient use of reagents, but at the same time maintaining sequence quality. Secondly, reduction of the sequencing primers. Most protocols use six to eight primers to sequence the poor region covering the protein gene in the first third of the reverse transcript gene, a set of four primers or selected to sequence the same region without compromising on the quality of the final sequence, thereby reducing the sequencing stage caused by at least a third.
Thirdly, the use of mostly free open access software and programs for skills analysis and report generation allowed for a further reduction in cost. And lastly, collective bargaining and public-private partnerships. A discounted price was negotiated that allows members of the Southern African Treatment and Resistance Network to get discounted rates for all the components of this protocol.
These have also been concerned to a kit for his access.
