Norovirus real-time fluorescent RT-PCR detection kit and application thereof

A technology of RT-PCR and kit, which is applied in the field of real-time fluorescent RT-PCR detection kits for type I and/or type II norovirus, and can solve the problem of low sensitivity

Inactive Publication Date: 2013-06-05
湖北朗德医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (1) Electron microscopy: Electron microscopy includes direct electron microscopy (EM) and immunoelectron microscopy (IEM). Direct electron microscopy requires at least about 10 5 ~10 6 virus particles, the sensitivity is low, and the detection rate is only 10% to 20% when patients excrete a large amount of virus in the early stage.

Method used

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  • Norovirus real-time fluorescent RT-PCR detection kit and application thereof
  • Norovirus real-time fluorescent RT-PCR detection kit and application thereof
  • Norovirus real-time fluorescent RT-PCR detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Development of norovirus one-step real-time fluorescent quantitative RT-PCR reagent

[0043] 1. Design of primers and probes: By using DNAman software to perform sequence comparison analysis on the existing Norovirus nucleic acid sequences in the Genebank database, the conserved fragment of the ORF1-ORF2 junction region of the Norovirus genome was used as the amplification target site According to the basic principles of primer-probe design, use software to manually design multiple pairs of primers and probes.

[0044] 2. Selection of samples: According to relevant domestic and foreign literature reports, you can choose samples from stool samples, anal samples, extracts containing stool or anal samples, or culture supernatants, such as stools, anal swabs, etc.

[0045] 3. Establishment and optimization of the reaction system

[0046]Sample preparation: 10 samples that were positive for Norovirus type Ⅰ were used as NoV Ⅰ positive reference samples, namely No...

Embodiment 2

[0055] Embodiment 2: Norovirus one-step fluorescent real-time quantitative RT-PCR detection kit and its use

[0056] 1. Prepare a kit including the following components: RNA extraction solution, RT-PCR amplification reaction solution, negative quality control substance, positive quality control substance, norovirus positive standard substance (quantitative reference substance), DEPC treated water.

[0057] 2. Collection, transportation and preservation of specimens

[0058] 2.1 Applicable specimen types: stool, anal swab, etc.

[0059] 2.2 Specimen collection and pretreatment (pay attention to aseptic operation)

[0060] 2.2.1 Stool specimen collection: Stool collection should be carried out by specially trained personnel. The specimen collector collects 5g (5ml) of stool and puts it in a sterile stool sampling cup (without adding any reagents).

[0061] 2.2.2 Anal swab specimen collection: soak cotton swab in normal saline, insert it into the anus 2-3cm, wipe it from the fo...

Embodiment 3

[0075] Example 3: Norovirus one-step fluorescence real-time quantitative RT-PCR detection kit for clinical detection

[0076] The above method was used to detect 68 stool samples of other patients suspected of norovirus infection, and the fluorescent quantitative PCR was set to the FAM / VIC dual-channel detection mode, which can detect NoV I and NoV II viruses at the same time. Among them, NoV I detection results were positive in 4 cases, the fluorescent probe for NoV I detection was FAM marker, and the FAM channel was selected for fluorescent quantitative PCR to analyze the results. The amplification curve is shown in Figure 5 , according to the C of these 4 positive results t Values ​​combined with amplification curves were automatically analyzed by RocheLightCycler480 analysis software to obtain the virus concentrations of these 4 cases of NoV I-positive samples. The specific results are shown in Table 1. Among them, NoV II detection results were positive in 5 cases, and t...

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Abstract

The invention relates to a norovirus real-time fluorescent RT-PCR detection kit and an application thereof. The invention belongs to the field of gene detection. The kit provided by the invention comprises a pair of oligonucleotide primers and an oligonucleotide probe aiming at type I norovirus obtained by screening, and/or a pair of oligonucleotide primers and an oligonucleotide probe aiming at type II norovirus obtained by screening. With a one-step real-time fluorescent RT-PCR, detectable minimal concentration of type I and/or type II norovirus is 1.0*10<2>copies/mL. Therefore, sensitivity and specificity of the kit provided by the invention are both high. With the invention, rapid early-stage detection and quantitative analysis of norovirus in samples such as stools and rectal swabs are realized. With the kit provided by the invention, detection period is short, detection efficiency is high, virus detection specificity is high, accuracy is high, and virus qualitative analysis and quantitative analysis can be carried out simultaneously. The sensitivity of the kit is higher than common PCR and immunological detection methods. The operation is simple, and the kit is easy to popularize. Experiment result repeatability is good.

Description

technical field [0001] The invention belongs to the field of gene detection, and relates to a type I and / or type II norovirus real-time fluorescent RT-PCR detection kit and application thereof. The kit of the present invention contains a pair of oligonucleotide primers and an oligonucleotide probe against Type I Norovirus obtained by screening, and / or a pair of oligonucleotides against Type II Norovirus Primers and an oligonucleotide probe, the minimum concentration of type I and type II norovirus that can be detected by one-step real-time fluorescent RT-PCR is 1.0×10 2 copies / mL, which shows that the sensitivity and specificity of this kit are very high. Background technique [0002] Norovirus (Norovirus, NV) is an American scholar Kapikian in 1972 through immunoelectron microscopy technology, first found in the feces of patients with diarrhea outbreaks in Norwalk Town, Ohio, USA, so it was first called Norovirus ( Norwalk virus). Since then, researchers have continuousl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 石康王琳琳朱世新
Owner 湖北朗德医疗科技有限公司
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