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Method for simultaneously detecting multiple diarrhea viruses by utilizing melting curve

A diarrhea virus, melting curve technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of detection accuracy not as good as nucleic acid detection, inability to achieve high-throughput detection, etc.

Pending Publication Date: 2021-03-30
JIANGSU BIOPERFECTUS TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, immunochromatographic detection is the most common method, but its detection accuracy is far inferior to nucleic acid detection, and nucleic acid detection cannot achieve high-throughput detection due to the influence of the number of detected fluorescence.

Method used

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  • Method for simultaneously detecting multiple diarrhea viruses by utilizing melting curve
  • Method for simultaneously detecting multiple diarrhea viruses by utilizing melting curve
  • Method for simultaneously detecting multiple diarrhea viruses by utilizing melting curve

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Embodiment 1: primer, probe design:

[0129] Search rovA, rovB, rovC, GI, GII, asv, sav, eadv, human ribonucleotidase P gene sequences through NCBI, compare the sequences through BioEdit, and use Primer Express 3.0 according to the technical principle described in 3.2.1 .1 Design primers and probes, confirm the specificity of primers and probes by BLAST, and obtain primers 1, 2, and probes corresponding to internal reference gene primers and eight pathogens.

[0130] Specific primers and probes required by the present invention include:

[0131] (1) Primer probes for amplifying rovA, the preferred final concentrations of primer probes are 300nM, 300nM, and 100nM respectively, and the sequences are as follows:

[0132] RovA-F: 5'-TTGGACCWTCTGATTCTGC-3',

[0133] RovA-R: 5'- GGTTAAGATAAATGC GAATCCATAGACAC-3',

[0134] RovA-P: (VIC fluorescein labeled): 5'-VIC-TCTGCATTTGTCTTAACCGCAT-BHQ1-3',

[0135] (2) Primer probes for amplifying rovB, the preferred final concentra...

Embodiment 2

[0167] Embodiment 2: the extraction of template nucleic acid:

[0168] (1) The collection of samples, including the collection of stool samples, the specific collection method can refer to the collection method of stool samples in the clinical laboratory center

[0169] (2) Extract the nucleic acid from the above collected samples according to the instructions of the nucleic acid extraction kit. The following extraction kits can be used: Viral Nucleic Acid Extraction Kit (Cat. No.: SDK60104, SDK60105) from Shuoshi Biological Company.

Embodiment 3

[0170] Embodiment 3: the preparation of primer probe detection solution and reaction solution:

[0171] (1) Preparation of primer probe detection solution:

[0172] .

[0173] (3) Preparation of reaction solution:

[0174] .

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Abstract

The invention discloses a method for simultaneously detecting multiple diarrhea viruses by utilizing a melting curve. The method comprises the following steps: designing eight groups of specific primer probes according to gene conservative fragments of viruses; designing a pair of reference gene primer probes EF, ER and EP according to the human ribonucleotidase P gene; combining all the primer probes and materials required by PCR into a reaction solution; adding sample nucleic acid into the reaction solution, and amplifying on a fluorescent PCR instrument by using a PCR amplification technology in combination with melting curve analysis; and judging whether the sample is positive or not according to the fluorescence amplification signals of different channels, and typing the positive nucleic acid in the corresponding fluorescence channels by adopting a melting curve analysis result.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a method for simultaneously detecting multiple diarrhea viruses by using melting curves. Background technique [0002] Diarrhea is a common gastrointestinal symptom that can occur all year round. Most of the pathogens causing diarrhea in my country are rotavirus, norovirus, astrovirus and saruvirus. Norovirus is one of the main pathogens causing viral diarrhea in the world. It contains 5 genotypes, among which the ones that infect humans mainly include type I and type II; Zarovirus is the most common virus that causes viral acute gastroenteritis One of them usually causes mild gastroenteritis among infants; Astrovirus has been confirmed to be one of the important pathogens causing diarrhea in infants, the elderly, and immunocompromised persons; and type 41 are one of the main pathogens causing viral diarrhea in children, and the course of the disease g...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2561/113C12Q2563/107C12Q2527/107
Inventor 李伟伟赵西浩曹新文王政峰张梅梅李秀林张蓉王河清贾兆强刘中华王国强
Owner JIANGSU BIOPERFECTUS TECH CO LTD
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