I, II herpes simplex virus fluorescence quantitative PCR detection method and kit thereof

A technology of herpes simplex virus and detection method, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. The effect of good sensitivity

Inactive Publication Date: 2010-03-24
YANGTZE RIVER PHARMA GRP BEIJING HAIYAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] (1) Although the isolation and culture method is the gold standard meth

Method used

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  • I, II herpes simplex virus fluorescence quantitative PCR detection method and kit thereof
  • I, II herpes simplex virus fluorescence quantitative PCR detection method and kit thereof
  • I, II herpes simplex virus fluorescence quantitative PCR detection method and kit thereof

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Effect test

Embodiment 1----I、I

[0053] Embodiment 1---I, type II herpes simplex virus fluorescent quantitative PCR detection method and its kit

[0054] Select primers and probes, and extract genomic DNA from the culture of the attenuated herpes simplex virus strain to be tested by alkaline thermal lysis. Specific steps are as follows:

[0055] 1) Add 1ml of sterile saline to the test tube of the specimen, shake well;

[0056] 2) Transfer the liquid to a 1.5ml centrifuge tube, centrifuge at 12,000rpm for 5 minutes and repeat;

[0057] 3) Add 40ul nucleic acid extraction solution (DNA) directly to the precipitation and mix thoroughly (the extract solution needs to be dissolved at room temperature and fully mixed before use);

[0058] 4) Boiling water bath for 10 minutes, turn to 4°C and let stand for 5 minutes;

[0059] 5) Centrifuge at 12,000rpm for 3 minutes;

[0060] 6) Take 2ul of the supernatant for PCR reaction.

[0061] In the 50 μl fluorescent PCR reaction system, add 2 μl of the herpes simplex v...

Embodiment 2

[0062] Embodiment 2---clinical testing

[0063] Using the above method to detect 115 cases of clinical samples, including 42 cases of herpes simplex patients, the positive rate of detection is 99%, the accuracy rate is 98.6%, and it can accurately quantitatively analyze the types of herpes simplex virus I and II, which is far superior to enzyme Linked immunosorbent assay (ELISA) method. The invention has the advantages of good specificity, high sensitivity, accurate quantification, rapidity, convenience and high repeatability, and can quickly and quantitatively detect the herpes simplex virus. In addition, the detection method of herpes simplex virus type I and II fluorescent quantitative PCR reduces the workload and cost of PCR diagnosis of clinical samples, and has potential application value.

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Abstract

The invention relates to an I, II herpes simplex virus fluorescence quantitative PCR detection method and kit thereof. Primers are designed direct towards DNA enzyme highly conservative region and then processed by PCR and a fluorescent probe is designed in the middle of the amplification sequence and then the annealing temperature of the reaction and the concentrations of the primers, the probe,the magnesium ion are optimized to establish the I, II herpes simplex virus real-time fluorescence PCR detection method. The kit ensures the sensitivity and accuracy and has features of simple and quick operation, reduced labor intensity of clinical detection, reduced cost of clinical PCR diagnosis, quick and simple performance of fluorescence quantitative PCR detection method. The method is usedfor quantitative detection of I, II herpes simplex virus with practical clinical application value.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection method and a kit for rapidly quantitatively detecting serum DNA of different genotyping herpes simplex virus I and II (herpessimple virus, HSV I, II) types, and belongs to the field of biotechnology. Background technique [0002] Herpes simplex virus is also one of the common sexually transmitted diseases in my country. The incidence of this disease is high. It can infect newborns through the placenta and birth canal, leading to miscarriage and death of newborns. It is also related to the occurrence of cervical cancer. Specific therapy has been paid attention to by people. In foreign countries, the incidence of genital herpes is second only to gonorrhea and syphilis among sexually transmitted diseases, ranking third in the incidence of sexually transmitted diseases and ranking first among sexually transmitted diseases caused by viruses. Herpes simplex virus (HSV) belongs to the herpesvir...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 李晓雯唐先兵石和鹏肖静郑宜婷
Owner YANGTZE RIVER PHARMA GRP BEIJING HAIYAN PHARMA
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