Establishment and application of new HIV-1 drug resistance detection method

Abstract

The invention discloses the establishment and application of a novel HIV‑1 drug resistance detection method. The novel HIV‑1 drug resistance detection method established by the present invention has the advantages of: ① using multiple sets of primer combinations, taking into account the problem of low amplification efficiency of the target gene caused by sequence differences of different subtype strains; ② in one reaction tube The cDNA synthesis of PR, RT and IN target genes and the first round of nested PCR amplification of the target gene were completed, avoiding the need to amplify the target gene separately, simplifying the experimental process, and saving half of the reagent cost. The detection method established by the present invention can cover multiple strain subtypes and multiple gene regions at the same time, so that the efficiency of HIV-1 drug resistance detection is greatly improved, and has a significant impact on AIDS prevention and public health.

Description

technical field [0001] The invention relates to the establishment and application of a novel HIV-1 drug resistance detection method. Background technique [0002] Since the discovery of AIDS in 1981, HIV (Human immunodeficiency virus, HIV), the culprit of the disease, has spread rapidly around the world, infecting 37 million people successively, causing serious public health problems. In order to effectively control the rapid progress and spread of this disease, antiretroviral therapy (ART) is used as a measure to control the course of the individual disease, and also as a means to manage the source of infection for the prevention of HIV infection. There are currently 2,009 million infected patients received ART. Judging from the treatment effects in 13 Asian countries / regions, ART has played an important role in immune reconstruction. CD4 cells can rise from a low point of 115 / μL to 302 / μL, and the virus suppression rate can reach 80 in some areas. %about. China currentl...

AI Technical Summary

Problems solved by technology

However, the disadvantages of using this method are: ① The amplified gene fragment is relatively long (about ~ 3153bp), so the efficiency in cDNA synthesis and target gene amplification is low (positive rate is about 65%), so it cannot meet the needs of clinical treatment. The need for drug resistance detection; ② The determination of the target gene sequence requires multiple reactions to complete

In order to ensure the accuracy and reliability of HIV drug resistance detection results, about 8 positive and negative primers are required to complete the bidirectional coverage sequence determination of this region to complete the evaluation of drug resistance results. Therefore, sequencing is difficult and expensive; ③HIV RT drug targets are mainly concentrated before the 348th position of the amino acid codon, and the impact of the more than 200 codons on the inhibitor is not very clear, so it is not used as the main site for judging drug resistance, but this region is amplified when the entire gene sequence of the pol region is amplified Amplified and sequenced at the same time, resulting in a certain degree of waste; ④The currently used drug resistance comparison database is the HIV drug resistance database of Stanford University (http: / / hivdb.stanford.edu / ), the data is accurate to the length of the submitted sequence There are certain requirements (<3000bp), so the length of the comparison of PR, RT and IN-related drug resistance sites using the full-length sequence of the pol region is limited

Amplification and detection of different inhibitor drug targets separately is a popular scheme at present. The disadvantages of this scheme are: ① multiple segments and multiple cDNA synthesis are required, and the price and cost are high; The amount of test samples and nucleic acid purification reagents requires a large amount

Images