This invention provides the application of primer sets in the preparation of reagents for HIV-1
gene drug resistance detection based on next-generation sequencing technology, and a kit thereof. The reagents are used to detect
drug resistance mutations in the HIV-1
gene, including subtypes A1, B, C, D, CRF01_AE, CRF07_BC, CRF08_BC, and CRF55_01B. The primer sets generate
amplicon lengths of 220-280 bp, and the
nucleotide sequences of the primer sets are shown in SEQ ID NO. 1-40. This invention employs a short
amplicon enrichment combined with two-round PCR
library construction, splicing the full-length pol
gene sequence through
amplicon overlap regions. Compared to in-house methods based on long fragment amplification, short amplicon methods offer higher amplification stability and detection sensitivity for samples with low viral loads and
nucleic acid degradation. Meanwhile, compared to long amplicon fragmentation
library construction, this method directly obtains the next-generation sequencing
library through two rounds of PCR operations. The experimental procedure is simple and can be completed in just 3.5 hours.