Primer pair, probe and kit used for quantitative detection total DNAs of HIV-1 and capable of covering multiple subtypes of HIV-1

An HIV-1, detection kit technology, applied in recombinant DNA technology, microbial assay/test, DNA/RNA fragment, etc., can solve problems such as high cost, lack of clinical data on genotype and subtype coverage, and cumbersome operations. High sensitivity and specificity, overcoming tedious and cost-increasing effects

Active Publication Date: 2018-12-14
BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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AI Technical Summary

Problems solved by technology

Due to the large number of amplification primer pairs, the biggest technical problems are cumbersome operation, high cost, amplification contamination and inaccuracy and other practical application ...

Method used

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  • Primer pair, probe and kit used for quantitative detection total DNAs of HIV-1 and capable of covering multiple subtypes of HIV-1
  • Primer pair, probe and kit used for quantitative detection total DNAs of HIV-1 and capable of covering multiple subtypes of HIV-1
  • Primer pair, probe and kit used for quantitative detection total DNAs of HIV-1 and capable of covering multiple subtypes of HIV-1

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Embodiment 1

[0042] Embodiment 1 Construction of the detection kit of the present invention

[0043] (1) Construction of HIV DNA Quantitative Standards

[0044] The HIV gene and part of the CD3 internal reference gene were respectively connected to the cloning vector by T-A cloning, and the two genes were connected in series to the same plasmid vector through enzyme digestion and ligation reactions to construct a standard containing both the HIV target gene and the internal reference gene.

[0045] The specific implementation is as follows: the amplified fragment of HIV LTR gene 554bp (nt356~nt890, HXB2) and the amplified fragment of human CD3 gene 812bp (nt460~nt1251, NC_000011.10) were simultaneously connected to the same pMD20- On the T vector vector (Takara, Code No.6028), positive plasmids containing two target gene fragments were identified by PCR, enzyme digestion and ligation. Extract the plasmid and measure its concentration, and calculate the plasmid copy number according to the...

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Abstract

The invention discloses a primer pair, probe and kit used for quantitative detection total DNAs of HIV-1 and capable of covering multiple subtypes of HIV-1. The sequence of the upstream primer HIV-U1of the primer pair is as shown in SEQ ID No. 1; the sequence of the downstream primer HIV-R1 of the primer pair is as shown in SEQ ID No. 2; the sequence of the probe Probe-HIV is as shown in SEQ ID No. 3. According to the invention, degenerate primers are utilized for high-sensitivity high-specificity detection of HIV DNA, and 12 common subtypes of HIV and rare HIV subtypes can be successfully detected, so the detection kit of the invention has wider subtype detection coverage compared with other conventional commercial detection kits. The primer pair, probe and kit provided by the inventioncan be used for quantification of total HIV DNAs of current prevalent HIV strains in China. A method provided by the invention is simple to operate, can complete procedures from sample DNA extractionto real-time fluorescent quantitative PCR amplification in one day, has low price, can be used for monitoring the persistent infection status of HIV-infected individuals in large cohorts, evaluating the effect of cART treatment, and is also applicable to auxiliary diagnosis of early HIV infection and diagnosis of neonatal infection in HIV-positive mothers.

Description

technical field [0001] The invention relates to a HIV-1 total DNA quantitative detection primer pair, a probe and a detection kit, specifically a highly sensitive and specific HIV-1 total DNA quantitative detection primer pair that can cover multiple subtypes, Probes and detection kits. Background technique [0002] As of the end of May 2018, my country reported 810,000 living HIV / AIDS patients and 250,000 reported deaths. There are still 30% of infected people who are not aware of the infection and have not been discovered. The epidemic situation is not optimistic. Therefore, early detection of HIV-infected persons, early treatment, reducing the viral load of infected persons, and controlling the source of infection from the source are an important strategic measure for the prevention and control of HIV infection. [0003] After HIV infection, the first viral markers to appear are viral nucleic acids and antigens. After the HIV virus enters the human body, it first infect...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/703C12Q2537/143C12Q2545/114
Inventor 刘志英袁霖粟斌张彤吴昊刘利锋陆小凡张欣王蕊夏炜黄晓婕
Owner BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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