Nucleic acid amplification fluorescent quantitation method for detecting and integrating HIV-1 virus genomes and application thereof

Abstract

The invention provides a nucleic acid amplification fluorescent quantitation method for detecting and integrating an HIV-1 virus genomes and application thereof. The method comprises the following steps of (1) building a real-time absolute quantitative HIV total DNA method; (2) preparing a standard product used for integrating an HIV genome; using the method in the step (1) for quantification; (3)performing gradient dilution for integrating an HIV standard product; performing nested fluorescence-quantitative PCR for building an HIV integration standard curve; (4) simultaneously preparing an HIV integration standard product and a sample to be tested; installing an Alu-primer-free reference pipe for each sample to be tested; after the PCR operation in the step (3), comparing the amplification result of the same to be tested with the standard curve to obtain the absolute copy number of the sample to be tested, wherein the virus copy number of the sample to be tested before the HIV integration equals to the copy number difference of the Alu primer sample and the Alu-primer-free reference pipes. The method can be used for distinguishing the integrated and non-integrated HIV virus genome; the virus before the specificity detection has very high sensitivity (5 to 10 copy / mul); the technical guarantee is provided for accurately evaluating HAART treatment effect and precisely quantifying the virus storage base in bodies of HIV patients.

Description

technical field [0001] The invention relates to the field of diagnosis, in particular to a nucleic acid amplification fluorescence quantitative method for detecting integrated HIV-1 virus genome and its application. Background technique [0002] AIDS is an infectious disease caused by human immunodeficiency virus type 1 (Human Immunodeficiency Virus 1, HIV-1) infection, which seriously threatens the safety of human life. Ten thousand people died, and there are still 37 million HIV-infected people in the world. [0003] The first prerequisite for containment and prevention of AIDS is rapid and efficient diagnosis and testing. There are more than 100 methods for detecting HIV at present, which are generally divided into serological diagnosis and virological diagnosis. Serological detection is the main method for early HIV diagnosis, and HIV infection is indirectly diagnosed by detecting HIV antibodies. Enzyme-linked immunoassay (ELISA), as a main means of AIDS diagnosis, ha...

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Problems solved by technology

However, there are various forms of HIV-1 DNA in virus-infected cells, including linear non-integrated, 1-LTR circular, 2-LTR circular and integrated, among which integrated HIV-1 DNA only accounts for the total DNA A very small part (up to 1 / 25), and more importantly, the integrated HIV virus genome has high transcriptional activity and is the main form of virus storage. Once the drug is stopped, the virus will rebound quickly from the storage , and currently many literature reports only use the real-time PCR method to quantify HIV-1 total DNA and use it to determine the content of the virus reservoir, so it is inaccurate

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