Kit for detecting HSV-2 nucleic acid

A technology of HSV-2 and HSV2-F, which is applied in the field of molecular biology, can solve the problems of low resolution and large fluorescence background noise, achieve high resolution, broad market application potential, and increase hybridization stability and specificity Effect

Inactive Publication Date: 2019-04-12
ZHEJIANG PROVINCIAL PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art, especially the quenching groups (such as Dabcyl, BHQ1, BHQ2) that currently use Taqman probes will emit light, resulting in large fluorescence background noise and low resolution. The main features of the present invention It is a non-luminescent minor groove binder (such as MGB) that is labeled at the 3' end of the detection probe, providing a Taqman-MGB for rapid detection, good specificity, and high resolution of HSV-2 detection of herpes simplex virus type 2 nucleic acid Probes, primers, kits and detection methods to meet the needs of rapid and accurate detection

Method used

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  • Kit for detecting HSV-2 nucleic acid
  • Kit for detecting HSV-2 nucleic acid
  • Kit for detecting HSV-2 nucleic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the design of target fragment, primer and probe

[0029] 1. Search the virion glycoprotein G (gG) gene sequence of herpes simplex virus type 2 HSV-2 (Gene Bank ID is Z86099.2) through the NCBI database, and screen out herpes simplex virus 2 through homology comparison verification of the gG gene Type HSV-2-specific gG gene target sequence.

[0030] 2. Using Primer Express 3.0 software, specific primer pairs and specific probes were designed for the target sequence of the herpes simplex virus type 2 specific gG gene. The 5' end of the probe is labeled with reporter fluorescein or fluorescent dye (in this example, FAM), and the 3' end is labeled with a minor groove binder (in this example, MGB). The target gene sequence, primers and probes were all synthesized by Hangzhou Qingke Zixi Biotechnology Co., Ltd., the primers and probes were purified by HPLC, and the freeze-dried powder was stored at -80°C. Perform quality inspection analysis on specific primer ...

Embodiment 2

[0036] Embodiment 2: PCR reaction solution, herpes simplex virus type 2 positive quality control substance and negative quality control substance

[0037] 1. The PCR reaction solution includes Taq enzyme, dNTP, buffer and other components. You can use the PCR reaction solution purchased from Hangzhou Qingke Zixi Biotechnology Co., Ltd., such as (TSE301) 2 xT5 Fast qPCR Mix (Probe).

[0038] 2. Preparation of herpes simplex virus type 2 HSV-2 positive quality control product: using herpes simplex virus type 2 HSV-2 specific primers to amplify the artificially synthesized target gene sequence (SEQ ID NO.1), through genetic engineering methods Construct the PUC57-HSV2-gG recombinant plasmid, transduce it into Top10 bacteria for plasmid amplification and extract the plasmid, and this plasmid solution is the herpes simplex virus type 2 HSV-2 positive quality control product. The schematic diagram of the pUC57 plasmid vector used to construct the MD19-T-HSV2-gG recombinant plasmid i...

Embodiment 3

[0041] Embodiment 3: detection method

[0042] 1. Template DNA preparation: wash the secretion swab with 1 ml of sterile normal saline, transfer all the washing liquid to a 1.5 mL centrifuge tube, and centrifuge at 12000 rpm for 5 min; remove the supernatant, take the precipitate and add 50 μL ddH 2 0 to mix well, 100 °C constant temperature treatment for 10 min; centrifuge at 12000 rpm for 5 min, and take the supernatant as a template for later use.

[0043] 2. Preparation of standard curve: Herpes simplex virus type 2 HSV-2 positive quality control product, diluted to 1ng / μL, and then 10-fold serial dilution to obtain 6 concentration gradients S1-6, namely 1ng / μL, 10 -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL. Using HSV2-P, HSV2-F and HSV2-R to detect S1-6 in real-time fluorescent PCR on ABI7500 and generate a standard curve, and analyze the amplified product by gel electrophoresis, the results are as follows Figure 8 , Figure 9 with Figure 10 sh...

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Abstract

The invention discloses a kit for detecting HSV-2 nucleic acid, comprising a specific primer pair HSV2-F and HSV2-R for detecting herpes simplex virus type 2, a fluorescent probe HSV2-P and a positivequality control substance. Both ends of the probe are modified separately, the 5' end is labeled with report fluorescein or fluorescent dye, and the 3' end is labeled with any minor groove binder without a fluorescent background. The positive quality control substance is a solution of a recombinant PUC57 plasmid containing a herpes simplex virus type 2 HSV-2 target gene gG sequence. The kit of the invention has the characteristics of fast and accurate detection, high resolution, high specificity, high efficiency and low cost, can realize qualitative detection of herpes simplex virus type 2 HSV-2, and has wide clinical application values.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a Taqman-MGB probe, primer, kit and detection method for specifically detecting herpes simplex virus type 2 nucleic acid, which are suitable for qualitative and quantitative detection of herpes simplex virus type 2. Background technique [0002] Herpes simplex virus (HSV) belongs to the genus Alphaherpesviridae and is a linear double-stranded DNA virus. Humans are the only natural host of herpes simplex virus. It is divided into two serotypes, namely HSV-1 and HSV- 2. The homology of the two genome sequences is about 50%. HSV has a wide range of infection sites, but the two subtypes of HSV are quite different in clinical manifestations, transmission routes and epidemiological characteristics. HSV-1 mainly causes febrile herpes and ulcers on the skin other than the genitals, oral mucosa, tonsils, and nasal vestibule, as well as eye keratitis. HSV-2, on the other hand...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/705C12Q2561/101
Inventor 葛玉梅赵钊
Owner ZHEJIANG PROVINCIAL PEOPLES HOSPITAL
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