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Deep sequencing-based detection method suitable for inferior drug-resistant strain of HIV-1 protease

A HIV-1PR and drug-resistant technology, applied in the biological field, can solve the problems of single detection site, high detection cost, and lack of comprehensive promotion of detection

Active Publication Date: 2020-08-21
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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  • Deep sequencing-based detection method suitable for inferior drug-resistant strain of HIV-1 protease
  • Deep sequencing-based detection method suitable for inferior drug-resistant strain of HIV-1 protease
  • Deep sequencing-based detection method suitable for inferior drug-resistant strain of HIV-1 protease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1. Establishment of a deep sequencing method suitable for HIV-1 PR 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 serially diluted to a solution with a concentration of 20 μM. Mix the diluted primers, among which 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, and mix them thoroughly for later use, and name them as primer combination-IV.

[0091] (2) Preparation of primers for deep sequencing in the second round of detection

[0092] Use ddH for the primers in Table 2 and Table 3 2 O was diluted to a solution with a concentration of 20 μM, mixed equal volumes of the primers in Table 2 and Table 3 according to the numbers of A01, B01, C01... in...

Embodiment 2

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

[0103] 1. Experimental samples

[0104] The plasma samples of 72 HIV-infected patients newly diagnosed and about to receive antiviral treatment (all signed the informed consent form) 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 drug resistance in PR region genes of HIV-1 infected patients

[0106] The plasma of each patient in step 1 was subjected to the following experiments:

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

[0108] All steps were performed in accordance with the instruction manual of MagNA Pure LC Total Nucleic Acid Isolation 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 in step 1 as a template,...

Embodiment 3

[0123] Embodiment 3, detection system specific detection

[0124] In order to verify whether the established detection system has cross-positive detection of 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, and the above-mentioned The testing process tests the samples. The detection of HCV samples completely followed the above-mentioned detection reaction system and procedures; in the first round of detection of HBV, reverse transcription at 50°C for 32 minutes was omitted, and the detection system and procedures were the same as before. The detection result shows that positive control CNHN24 detection result has good specificity, HBV, HCV detection sample and negative control (with ddH 2 O is the template) there is no specific target band, that is, the test result is negative. The results of specific detection confirmed that the established detectio...

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Abstract

The invention discloses a deep sequencing-based detection method suitable for an inferior drug-resistant strain of HIV-1 protease. The invention provides a primer group for HIV PR drug resistance detection. The primer group is composed of six primers as shown in SEQ ID No. 1-6 and two primers as shown after Barcode sequences are connected to the 5'-terminal of SEQ ID No. 7 and SEQ ID No. 8 respectively. The cDNA synthesis of the PR and the first round of amplification of the nested PCR are completed in the same reaction system; a Barcode sequence is added to the head and the tail of the primerin the second round of nested PCR amplification; and after the purification of the product of the second round of amplification, high-throughput sequencing is carried out to obtain HIV-1PR inferior drug-resistant strain information. The novel HIV-1 protease inferior drug-resistant strain drug resistance detection method established by the invention is low in cost and high in yield. The inventionhas great influence on AIDS prevention and treatment and public health.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting HIV-1 protease inferior drug-resistant strains based on deep sequencing. Background technique [0002] Since HIV was discovered in 1981, it has spread rapidly around the world, causing nearly 37 million people to be infected, and has caused 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 promoted and applied around the world as a prevention and control method. This method has effectively delayed the disease progression of HIV-infected patients and controlled the rapid spread of AIDS. . Up to now, more than 21 million HIV-infected people around the world have received ART, which has played a positive role in virus suppression and immune reconstruction in infected people. In recent years, China has rapidly expanded treatment. At present, more...

Claims

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Application Information

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Patent Type & Authority Applications(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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