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A deep sequencing-based method suitable for the detection of HIV-1 protease inferior drug-resistant strains

An HIV-1PR, virus strain technology, applied in the biological field, can solve the problems of high detection cost, increased virological inhibition failure, single detection site, etc., and achieve the effect of reducing detection cost and sequencing cost.

Active Publication Date: 2022-06-07
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the field of antiviral therapy, the discovery of inferior drug-resistant strains in the untreated population will increase the risk of treatment failure, even if the share of inferior drug-resistant strains is less than 1%, it will increase the risk of virological suppression failure by 2.5% times; more and more data prove that the failure of most antiviral treatment is related to the presence of inferior drug-resistant strains at baseline
Although the above methods have played a certain role in the prevention and control of AIDS, the detection of HIV inferior drug-resistant strains has not been fully promoted due to the limitations of the method. For example, some methods detect too single sites and cannot distinguish polymorphic sites well. factors such as ASPCR, OLA, GeneChipTM), high detection cost and time-consuming and laborious (such as SGS), narrow detection range (TyHRT, LiPA) and other factors limit the promotion and use of the method
Therefore, although there is consensus on the importance of inferior drug-resistant strains in AIDS prevention and control, their implementation is slow due to detection methodological problems

Method used

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  • A deep sequencing-based method suitable for the detection of HIV-1 protease inferior drug-resistant strains
  • A deep sequencing-based method suitable for the detection of HIV-1 protease inferior drug-resistant strains
  • A deep sequencing-based method suitable for the detection of HIV-1 protease inferior drug-resistant strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1. Establishment of a deep sequencing method suitable for HIV-1PR drug resistance detection

[0088] 1. Primer combination

[0089] (1) Preparation of primers for the first round of RT-PCR

[0090] Take the synthesized primers DR1-1-CNB, DR2-1-CNBC, PRTM-F1a, PRTM-F1b, DR1-2-CNB and DR2-2-CNBC (Table 4), use ddH 2 O was sequentially diluted to a concentration of 20 μM. Mix the diluted primers, wherein DR1-1-CNB, DR2-1-CNBC, PRTM-F1a, PRTM-F1b are mixed according to 1 / 8 of the total volume, DR1-2-CNB, DR2-2-CNBC Mix them according to 1 / 4 of the total volume, mix well after mixing, and name the primer combination-IV.

[0091] (2) The second round of detection is intended to be used for the preparation of primers for deep sequencing

[0092] Use ddH for the primers in Table 2 and Table 3 2 Dilute O to a solution with a concentration of 20 μM, mix the primers in Table 2 and Table 3 in equal volumes according to the number of A01, B01, C01... in the primer name,...

Embodiment 2

[0102] Example 2. Sensitivity evaluation of deep sequencing method for HIV-1PR drug resistance detection

[0103] 1. Experimental samples

[0104] Plasma samples from 72 newly diagnosed HIV-infected patients who were about to receive antiretroviral therapy (all signed informed consent forms) were used as experimental samples, and the viral load of the infected patients was ≥1000Compies / ml.

[0105] 2. Using the established deep sequencing method to detect the drug resistance of HIV-1 infected PR region genes

[0106] The following experiments were performed on the plasma of each patient in step 1:

[0107] 1. Nucleic acid purification of plasma samples to be tested

[0108] All steps were performed according to the instructions of the nucleic acid purification kit MagNA Pure LC Total Nucleic AcidIsolation Kit (Code No. 3038505001) of Roche Company, and will not be repeated here.

[0109] 2. Preparation of deep sequencing samples

[0110] (1) Using the total RNA of step 1 a...

Embodiment 3

[0123] Embodiment 3, detection system specificity detection

[0124] In order to verify whether the established detection system has cross-positive detection for similar viruses, HBV samples (representing human hepatitis B virus, 26 samples) and HCV (representing human hepatitis C virus, 40 samples) stored in the laboratory were taken. The testing process tests the sample. The detection of HCV samples completely followed the above detection reaction system and procedures; HBV reverse transcription at 50°C for 32 min was omitted in the first round of detection, and the detection system and procedures were the same as before. The test results show that the positive control CNHN24 test results have good specificity, HBV, HCV test samples and negative control (with ddH 2 O is the template) no specific target band appears, that is, the detection result is negative. The specific detection results confirmed that the established detection system had no cross-reaction to similar viru...

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Abstract

The invention discloses a method for detecting HIV‑1 protease inferior drug-resistant strains based on deep sequencing. The present invention provides a primer set for HIV PR drug resistance detection, consisting of six primers shown in SEQ ID No.1-6, and after connecting the Barcode sequence at the 5' end of SEQ ID No.7 and SEQ ID No.8 respectively Two primers are shown. In the present invention, the cDNA synthesis of PR and the first round of nested PCR amplification are completed in the same reaction system; in the second round of nested PCR amplification, the Barcode sequence is added to the head and tail of the primer; the second round of amplification product is purified and carried out High-throughput sequencing to obtain HIV‑1PR inferior drug-resistant strain information. The drug resistance detection method of the novel HIV-1 protease inferior drug-resistant strain established by the invention has low cost and high output. The invention has great impact on AIDS prevention and public health.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a deep sequencing-based detection method for HIV-1 protease inferior drug-resistant strains. Background technique [0002] Since the discovery of HIV in 1981, it has spread rapidly around the world, infecting nearly 37 million people and causing serious public health problems around the world. In response to the three 90% prevention and control goals proposed by WHO, antiretroviral therapy (ART) has been widely used as a prevention and control method around the world. . Up to now, more than 21 million HIV-infected people around the world have received ART, which has played a positive role in viral suppression and immune reconstruction in the infected people. China has rapidly expanded treatment in recent years. Currently, more than 500,000 people living with HIV have received free ART, which has contributed to improving the quality of life of AIDS patients, prolonging the life cycl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6848C12Q1/6869C12N15/11C12R1/93
CPCC12Q1/703C12Q1/6848C12Q1/6869C12Q2531/113C12Q2535/122
Inventor 李林李韩平李敬云刘永健王晓林李天一韩靖婉贾磊
Owner ACADEMY OF MILITARY MEDICAL SCI
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