Method and kit for detecting genotypes of herpes simplex virus

Abstract

The invention discloses a method and a kit for detecting genotypes of a herpes simplex virus (HSV). Primer probes designed on the basis of HSV DNA polymerase gene are adopted, type 1 and type 2 of the HSV are detected simultaneously in a single tube by TaqMan probe dual-wavelength fluorescent PCR technology, and the existence and the type of the HSV in a sample are judged through fluorescent signals and circulating threshold values of amplification templates with different wavelengths. In the kit, a manually constructed internal reference system is used for avoiding false negative of a detection result. The kit comprises nucleic acid extracting solution, PCR buffer solution, Taq enzyme, an internal reference, negative control and positive control; and two steps of sample treatment and amplification detection are adopted. The kit is easy and convenient to operate and high in sensitivity, can avoid false positive caused by PCR product nucleic acid pollution, can solve the problem of false negative caused by a PCR inhibitor in the sample, and can be widely applied to quick detection of the genotypes of the pathogen clinically.

Description

technical field [0001] The invention belongs to a method and a kit for genotyping herpes simplex virus by utilizing fluorescent PCR technology. Background technique [0002] Herpes simplex virus (Herpes Simplex Viruses, HSV) belongs to the herpes virus family, and the diameter of the virus particle is about 180nm, with double-stranded DNA. 2 types, and the genome sequencing of HSV-1 and HSV-2 has been completed. Clinically, HSV can cause herpes on the face and lips (facial herpes, such as "oral herpes"), or herpes on the genital area (genital herpes), etc., and HSV infections in other parts of the genitals are mostly caused by HSV-1 (accounting for 95% ), and the HSV infection of reproductive organs is mainly caused by HSV-2 type (accounting for 78%), but both types of HSV can infect these two parts, and both can infect newborns. Generally, HSV can infect newborns through the birth canal during delivery, causing neonatal herpes, skin, eye or oral infection, damage to the c...

AI Technical Summary

Problems solved by technology

The genotyping method of HSV usually adopts molecular biological methods, such as strand substitution reaction (SDA) and PCR technology. At present, there are commercialized HSV genotyping detection kits abroad for scientific research or clinical diagnosis, including BD in the United States. , Roche and QIAGEN of Germany’s HSV-1 / 2 genotyping detection kits, of which BD’s products use SDA technology, and the market share is not high; while Roche’s and QIAGEN’s products use FRET probe-based fluorescent PCR technology and Melting curve analysis method, each sample needs to add a melting curve analysis step after fluorescent PCR is completed, and genotype HSV according to the Tm value of the amplified product, which requires many steps and takes a long time for detection

In addition, there are literatures (Anderson TP et al, J ClinMicrobiol, 2003, 41(5): 2135-2137) reporting that the use of melting curves for HSV genotyping has the disadvantage that the characteristics of the melting curve are not obvious and the results cannot be judged

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