HIV drug resistance testing
["Collaborating Experts | M.S. Denggao Xu", "Cell Biology Liaoning Normal University"], ["Reviewed by | Dr. Lei Wang", "Biochemistry and Molecular Biology Zhejiang University"]
Summary
HIV drug resistance testing can be phenotypic or genotypic testing, genotypic testing is the most commonly used testing method, the method is mainly through the means of PCR and gene sequencing to obtain HIV drug resistance-related gene sequences, and to analyze the genome for drug resistance-related amino acid mutations.
Principle
The presence of drug-resistant variants was determined by amplifying HIV sample gene fragments (gag, env, and pol) by PCR, sequencing the amplified target fragments, and comparing the sequencing results.
Appliance
PCR for detection of gag, env and pol gene variants in HIV-infected AIDS patients for HIV prevention and treatment.
Operation method
HIV drug resistance testing - PCR method
Principle
The presence of drug-resistant variants was determined by amplifying HIV sample gene fragments (gag, env, and pol) by PCR, sequencing the amplified target fragments, and comparing the sequencing results.
Materials and Instruments
PCR instrument, PCR buffer, dNTP mixture
HIV cDNA, TaqDNA polymerase
Deionized water, DNA purification and recovery kit
PCR primers, electrophoresis
Move
The steps for detecting HIV drug resistance by PCR are as follows:
1. Design primers: Select the gene fragments to be amplified (gag, env and pol) and design primers;
Prepare sterile 10 μL, 200 μL, 1000 μL tips and PCR tubes;
3. Add 1 μL of 2.5 mM 4×dNTPs mixture, 5 μL of 10×PCR buffer, 5 μL of 25 μM MgCl2, 1 μL of the upstream primer (10 μM), 1 μL of the downstream primer (10 μM), 0.2 μL of TaqDNA polymerase, and 3 μL of the HIV cDNA, and top up the rest to 50 μL with deionized water. The remaining amount should be made up to 50 μL with deionized water, and then mix well;
4, set the first round of nested PCR reaction program: 95 ℃ pre-denaturation 3 min, 95 ℃ denaturation 30 s, 55 ℃ annealing 30 s, 72 ℃ extension for 1 min, denaturation, annealing, extension of the three operations to carry out 35 cycles, the final extension of 72 ℃ for 5 min, 4 ℃ storage;
5. Second round of nested PCR reaction: Add 1 μL of (2.5 mM) 4×dNTPs mixture, 5 μL of 10×PCR buffer, 5 μL of (25 μM) MgCl2, 1 μL of the upstream primer (10 μM), 1 μL of the downstream primer (10 μM), 0.2 μL of TaqDNA polymerase, and 5 μL of the product from the first round of nested PCR to the PCR tube. The remaining amount was made up to 50 μL with deionized water and mixed well;
6. The second round of the nested PCR reaction was programmed as follows: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min, 30 cycles of denaturation, annealing and extension, and a final extension at 72 ℃ for 5 min, and storage at 4 ℃;
7. The PCR products were electrophoresed on a 1% agarose gel. After identification, the products were purified and recovered by the DNA Gel Purification and Recovery Kit and used for PCR amplification and sequencing;
8. The PCR product from HIV is sequenced and compared with the wild-type sequence to determine whether there is a drug resistance-associated mutation.
Caveat
The primer binding site may appear multi-site binding, which may reduce the accuracy of the test; the experimental materials should be sterile to avoid contamination of the PCR reaction.
Common Problems
(1) Multiple bands appear in the electrophoresis product.
(2) Bad quality of template and primer.
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