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Fluorescent PCR kit for detecting herpes simplex virus type II

A herpes simplex virus and kit technology, which can be used in the determination/examination of microorganisms, biochemical equipment and methods, etc., can solve problems such as unfavorable HSV infection census and rapid diagnosis, unfavorable early diagnosis of HSV infection, and little clinical diagnostic value. , to reduce the risk of PCR product contamination, avoid reaction product contamination, and achieve the effect of fast detection

Inactive Publication Date: 2011-06-22
上海裕隆医学检验所股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although virus culture identification is the gold standard for the diagnosis of HSV infection, this method needs to isolate the herpes simplex virus and inoculate it into the tissue culture medium, and it takes 24 to 48 hours for the lesion to appear, which is time-consuming and has strict technical requirements, which is not conducive to General screening and rapid diagnosis of HSV infection; cytological examination of skin scrapings is to observe under a microscope whether there are multinucleated giant cells and eosinophilic inclusion bodies in the nucleus, etc. This method is for screening after clinical symptoms appear, which is not conducive Early diagnosis of HSV infection; serum HSV-IgM antibody detection, easy to detect false negative and false positive results, only applicable to epidemiological investigations, and has little value for clinical diagnosis

Method used

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  • Fluorescent PCR kit for detecting herpes simplex virus type II
  • Fluorescent PCR kit for detecting herpes simplex virus type II
  • Fluorescent PCR kit for detecting herpes simplex virus type II

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the preparation of kit

[0032] 1. Primer and probe design and synthesis

[0033] Use Primer Express 3.0 to screen the primers in the conserved region for the complete HSV II gene, and design HSV II probes based on the selected primers. Both primers and probes were synthesized by a professional company (Shenggong), wherein the primers were purified by PAGE, and the probes were purified by HPLC. The 5' end of the probe was labeled with a FAM fluorescent group, and the 3' end was labeled with a TAMRA fluorescent group.

[0034] The amplified sequence is shown in Table 1:

[0035] Table 1. Specific probe and primer sequences

[0036] sequence name

Oligonucleotide sequence (5'-3')

Base length (bp)

HSV II probe

tgctcatcaagggcgtggatctggtgc

27

HSV II forward primer

acgttcaccaagctgctgc

19

HSV II reverse primer

atcaaccgcacctccagg

18

[0037] 2. Preparation of HSV II plasmid positive templ...

Embodiment 2

[0045] Embodiment 2: the use of kit

[0046] 1. Sample extraction

[0047] Use the DNA extraction solution to extract the HSV II nucleic acid in the sample to be tested

[0048] 1) Sample pretreatment: wipe off excess secretions from the cervix, use a cotton swab soaked in normal saline to cling to the cervical mucous membrane and rotate it for 2 weeks to obtain secretions and exfoliated cells, and put the cotton swab after sampling Rinse fully in an EP tube with 1ml of sterile saline, and squeeze dry by sticking to the wall. The HSV II gene was extracted by PEG precipitation and alkaline lysis.

[0049] PEG precipitation solution formula: 30% PEG8000.

[0050] Lysis solution formula: 60mmol / L TrisHCl, pH 8.0; 0.5% SDS; 200mmol / L NaCl; 1M / L NaOH.

[0051] 2) Operation steps: Take 500 μl of secretion and an equal volume of PEG precipitation solution, shake and mix, centrifuge at 13,000 rpm for 10 minutes, discard the supernatant; add 50 μl of lysate, incubate at 100°C for 2...

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Abstract

The invention discloses a fluorescent polymerase chain reaction (PCR) kit for detecting herpes simplex virus type II, and belongs to the field of in-vitro diagnostic kit for nucleic acid. The kit comprises a positive reference, a negative reference, fluorescent polymerase chain reaction liquid, PCR primers and a specific fluorescent probe, polyethylene glycol (PEG) precipitation solution and lysis solution. The invention comprises a PCR system based on a fluorescent PCR technology, contains forward and reverse primers and the fluorescent probe for detecting the gene sequence of the herpes simplex virus type II, can detect the nucleotide sequence of the gene of the herpes simplex virus type II under proper PCR condition, can easily and quickly detect the infection of the HSV II in clinical samples and is high in specificity.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, in particular to a fluorescent PCR (polymerase chain reaction) kit for detecting herpes simplex virus type II (HSV-II) infection in clinical samples. Background technique [0002] Herpes simplex is an acute herpetic skin disease caused by herpes simplex virus (HSV). Human is the only natural host of herpes simplex virus. The virus exists in the blisters of patients, recoverers or healthy carriers. In scar fluid, saliva and feces, the main mode of transmission is direct contact infection, and it can also be indirectly transmitted through tableware contaminated by saliva. Human herpes simplex virus is divided into two types, namely herpes simplex virus type I (HSV-I) and herpes simplex virus type II (HSV-II). Type I mainly causes infections of the skin, mucous membranes (oral mucosa) and organs (brain) other than the genitals. Type II mainly causes skin and mucous membrane infection...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 穆海东汪宁梅穆宇豪黎飒
Owner 上海裕隆医学检验所股份有限公司
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