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

A technology for HIV-1 and drug resistance, applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of large amount of purification reagents, large amount of nucleic acid usage, waste, etc.

Active Publication Date: 2018-11-23
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
  • Description
  • Claims
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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 th...

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Embodiment 1, the primer combination that is used for HIV-1 drug resistance detection

[0113] Primer combination I: primer DR1-1-CNB, primer DR2-1-CNBC, primer DR1-2-CNB, primer DR2-2-CNBC, primer pol-1e, primer pol-x, primer IN1 respectively in Table 1 , Primer IN1-07BC, Primer IN2 and Primer IN2-07BC were configured into primer solutions of equal concentration using denucleic acid water, and then mixed in equal volumes to obtain primer combination I (the concentration of each primer in primer combination I was the same).

[0114] Primer Combination II: Prepare the primer DR-3, primer DR1-4-CNB and primer DR2-4-CNBC in Table 1 respectively to a primer solution of equal concentration using de-nucleic acid water, and then take 2 parts by volume of primer DR-3, 1 part by volume of primer DR1-4-CNB and 1 part by volume of primer DR2-4-CNBC are mixed to obtain primer combination II (concentrations of primer DR-3, primer DR1-4-CNB and primer DR2-4-CNBC in primer combination...

Embodiment 2

[0116] Embodiment 2, establishment of novel HIV-1 drug resistance detection method

[0117] 1. Extract the total RNA from the isolated plasma sample of the patient to be tested.

[0118] 2. Using the total RNA obtained in step 1 as a template, carry out the synthesis of cDNA and the first round of nested PCR amplification. The reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0119] Table 2 cDNA synthesis and the first round of nested PCR amplification system

[0120] composition

Volume (μL)

PrimeScript 1Step Enzyme Mix

1

2×1Step Buffer

12.5

Primer combination Ⅰ (the concentration of each primer in the system is 2μM)

1

RNase Free dH 2 o

5.5～0.5

RNA

5～10

total

25

[0121] Table 3 cDNA synthesis and the first round of nested PCR amplification reaction program

[0122]

[0123] 3. Carry out step (1) or step (2) according to the medication situation of the pati...

Embodiment 3

[0144] Embodiment 3, specificity

[0145] Five HTLV-infected patients with pre-clinical diagnosis and positive nucleic acid test (volunteers with informed consent; numbered HTLV25, HTLV26, HTLV36, HTLV37, HTLV38), and five HBV-infected patients with clinical diagnosis and positive nucleic acid test (informed consent) were used. Volunteers; numbered HBV-2, HBV-5, HBV-6, HBV-10, HBV-12), eight HCV-infected patients who were clinically confirmed and positive in nucleic acid tests (informed consent volunteers; numbered respectively HCV-5, HCV-70, HCV-71, HCV-75, HCV-76, HCV-93, HCV-7, HCV-56; where HCV-70 is the co-infection of HIV-1 and HCV infection) as The isolated plasma samples were used as experimental samples for specific detection.

[0146] The detection was carried out according to the method in Example 2. After cDNA synthesis and the first round of PCR amplification with primer combination I, the target gene was amplified again with primer combination II and primer combin...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6848C12N15/11
CPCC12Q1/6848C12Q1/6888C12Q2600/16C12Q2549/119C12Q2535/122C12Q2537/143
Inventor 李林李韩平李敬云刘永健韩靖婉贾磊李天一鲍作义王晓林刘思扬
Owner ACADEMY OF MILITARY MEDICAL SCI
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