HIV-1 Genotyping Kit Amplification Module

The Applied Biosystems™ HIV-1 Genotyping Kit is designed to genotype the protease and reverse transcriptase (RT) regions of the HIV-1 pol gene. The genotyping result encompasses codons 6-99 in the protease region and codons 1-251 in the RT region of the gene. HIV-1 genomic mutations in these regions confer resistance to anti-retroviral drugs that act through the inhibition of the protease and reverse transcriptase. The kit enables reliable genotyping of the genetically diverse HIV-1 virus to investigate resistance to protease inhibitors, nucleoside reverse-transcriptase inhibitors, and non-nucleoside reverse-transcriptase inhibitors from plasma and dried blood spot samples. This product employs assays for HIV-1 genotyping licensed from the U.S. Centers for Disease Control and Prevention.

Configuration
The HIV-1 Genotyping Kit is comprised of two modules: this Amplification module (Cat. No. A32317) and a Cycle Sequencing module (Cat. No. A32318). The Amplification module contains sufficient reagents for 48 RT-PCR reactions and 48 nested PCR reactions. The Cycle Sequencing module contains sufficient reagents for 48 sets of bidirectional cycle sequencing reactions. Positive controls are included in both modules.

Design
The HIV-1 Genotyping Kit harnesses the gold-standard Sanger sequencing technology to amplify and reliably sequence the diverse and rapidly evolving HIV-1 virus. Starting with viral RNA, the RT-PCR step amplifies a 1.3 kb region of the HIV-1 pol gene. The second, nested PCR generates a 1.1 kb amplicon from the RT-PCR product, which spans the entire protease gene and two-thirds of the RT gene. In the cycle sequencing step, three forward and three reverse sequencing primers provide full coverage of codons 6-99 in the protease region and codons 1-251 in the RT region.

Starting with viral RNA from a plasma sample or dried blood spot sample, the Amplification module generates a PCR product as DNA template. After confirming the band size of the PCR amplicon, the Cycle Sequencing module is used to sequence the DNA template utilizing Sanger sequencing technology. The sequencing reactions are first purified, then subjected to capillary electrophoresis. The sequencing results are used to create a mutation profile through a curated database, such as the HIV Drug Resistance Database from Stanford University. The mutation profile includes drug resistance information on protease inhibitors (PIs), nucleoside reverse-transcriptase inhibitors (NRTIs), and non-nucleoside reverse-transcriptase inhibitors (NNRTIs).

For more information, please email: HIV_GenotypingCustomerInquiry@thermofisher.com