Primer and kit for visual isothermal amplification detection of human immunodeficiency virus type I

Abstract

The invention belongs to the technical field of biomolecular detection, and relates to a visual isothermal amplification detection primer and a kit for human immunodeficiency virus type I. The invention designed the inner primers FIP and BIP, the outer primers F3 and B3 different from the existing primer sequences, and added two looping primers LoopF ​​and LoopB, which greatly shortened the time of nucleic acid amplification reaction. The optimal reaction temperature is constant temperature of 65°C, and the optimal reaction time is 30 minutes, which is at least half the time required for the original nucleic acid amplification reaction. At the same time, the 6 primers designed based on the present invention have a high degree of specificity, and the presence or absence of the target gene sequence can be judged only according to the presence or absence of the amplification reaction product, and nucleic acid detection can be quickly realized, fully satisfying the Current HIV‑1 nucleic acid testing needs.

Description

technical field [0001] The invention belongs to the technical field of biomolecular detection, and relates to a visual isothermal amplification detection primer and a kit for human immunodeficiency virus type I. Background technique [0002] Human immunodeficiency virus (Human Immunodeficiency Virus, HIV) is the main pathogen that induces human acquired immunodeficiency syndrome (Acquired Immunodeficiency Syndrome, AIDS). . [0003] In recent years, enzyme-linked immunosorbent assay (ELISA), PCR, cell culture and other methods are mainly used to detect HIV-1 antibody or virus at home and abroad. These methods have some disadvantages to varying degrees. [0004] For example, the ELISA method has complex operating procedures, which can easily cause false positives and false negatives, and easily cause harm to the operator and environmental pollution. The test time is relatively long, and it takes more than two hours to obtain the test result. At the same time, it is necessa...

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Problems solved by technology

[0004] For example, the operation procedure of ELISA method is complicated, it is easy to cause false positive and false negative, and it is easy to cause damage to the operator and environmental pollution. The test time is long, and it takes more than two hours to get the test result.

At the same time, it is necessary to have a microplate reader and a plate washer, which is difficult to achieve in grassroots laboratories and small outpatient clinics, and the test is limited by conditions such as temperature, which brings inconvenience to the test

[0005] Cell culture is a kind of aseptic operation technology, which requires high experimental working environment and conditions, such as sterile operation room, ultra-clean workbench, etc.); requires a large number of experimental equipment, such as incubator, centrifuge, microscope, etc.; A large amount of professional experimental equipment is required, and the experimental technology is complicated; experienced technicians are required to operate, and the experimental period is very long, which is not suitable for rapid detection of diseases

Agglutination tests including gelatin particles (PA) and latex particles (LAT) are semi-quantitative determinations, which require multiple dilutions of reagents, poor reproducibility of results, and low sensitivity

[0006] Although ordinary PCR technology has advantages in sensitivity and specificity, it also has several limitations in technical application: first, PCR technology requires expensive experimental instruments, and the instruments must have sophisticated temperature control procedures and heating templates, so as to realize The denaturation of the DNA template at a very high temperature, and the annealing of the primer probe at a lower temperature extends the template, so that the amplification of the template amount is achieved after repeating the temperature change for dozens of cycles

Second, because each cycle time is short, the instrument must be able to quickly and accurately raise and lower the temperature, which requires a silver or gold heating module, which increases the cost of instrument development

Excessive primers will mismatch with the template, misprime amplification, primer dimer, etc., thereby inhibiting PCR amplification, especially in the case of low initial template amount, which will exacerbate this phenomenon

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