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

Abstract

The invention discloses establishment and application of a novel HIV-1 drug resistance detection method. The novel HIV-1 drug resistance detection method established according to the invention has theadvantages that 1, by utilizing multiple sets of primer combinations, a problem of low amplification efficiency of target genes, which is caused by differences of different subtypes of strain sequences, is considered; and 2, synthesis of PR, RT and IN target gene cDNA and first-round amplification of a target gene nested PCR are completed in one reaction tube so as to avoid respective amplification on the target genes, simplify the experimental process and save reagent cost by 50%. The detection method established according to the invention can simultaneously cover multiple strain subtypes, and simultaneously cover multiple gene regions, which enables HIV-1 drug resistance detection efficiency to be greatly improved, and has the great influence 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

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