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Development of a simple microarray for genotyping HIV ‐1 drug resistance mutations in the reverse transcriptase gene in rural T anzania
Author(s) -
Masimba Pax,
Gare Janet,
Klimkait Thomas,
Tanner Marcel,
Felger Ingrid
Publication year - 2014
Publication title -
tropical medicine and international health
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.056
H-Index - 114
eISSN - 1365-3156
pISSN - 1360-2276
DOI - 10.1111/tmi.12289
Subject(s) - genotyping , sanger sequencing , reverse transcriptase , single nucleotide polymorphism , biology , snp genotyping , molecular inversion probe , genetics , population , genotype , variants of pcr , drug resistance , polymerase chain reaction , primer (cosmetics) , gene , microbiology and biotechnology , dna sequencing , medicine , chemistry , environmental health , organic chemistry
Objective Aiming at a simple, inexpensive and robust tool for HIV‐1 drug resistance genotyping during antiretroviral therapy (ART) we developed and validated a microarray‐based detection of 25 drug resistance mutations most relevant for the Tanzanian ART regimen. Methods A reverse transcriptase gene fragment was reverse‐transcribed and amplified by reverse transcription–polymerase chain reaction ( RT–PCR ). Primers for mini‐sequencing were designed based on alignments of the most prevalent local HIV ‐1 variants. Tagged primers were extended by fluorochrome‐labelled dideoxynuclotide triphosphate (dd NTP s) to indicate the single‐nucleotide polymorphism ( SNP ) allele of the sample tested, followed by hybridisation on treated microarray slides. Images were analysed with a laser scanner and genotype calling was performed using in‐house developed software. Results The microarray was validated with four cloned HIV ‐1 genome fragments from a Swiss HIV ‐1 cohort and 102 HIV ‐1 sequences amplified from the Tanzanian target population (field samples). Results were concordant with the Sanger sequencing SNP profile in 92.7% of 2550 SNP data points compared. Lack of signals in small number of SNP s was due to either failure in the extension reaction or hybridisation owing to mismatches between PCR product and extension primer. Conclusion Our study demonstrates the feasibility of hybridisation‐based genotyping of drug resistance mutations of HIV , even though our microarray, which was designed for population studies, achieved only correct assignment of 92% of all SNPs in the tested samples.