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Primer group and kit for simultaneously detecting four diarrhea viruses

A technology for detecting kits and diarrhea viruses, which is applied in the direction of microorganisms, recombinant DNA technology, and methods based on microorganisms. It can solve the problems of no HRMA analysis technology and no simultaneous detection, and achieve high sensitivity and specificity.

Inactive Publication Date: 2015-05-27
珠海国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no HRMA analysis technology for rotavirus and saruvirus, and there is no report on the development of HRMA technology for simultaneous detection of these four viruses

Method used

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  • Primer group and kit for simultaneously detecting four diarrhea viruses
  • Primer group and kit for simultaneously detecting four diarrhea viruses
  • Primer group and kit for simultaneously detecting four diarrhea viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Design of specific primers

[0043]First, download as many genomic DNA sequences of rotavirus, astrovirus, norovirus and saruvirus as possible from the NCBI gene bank in the United States, and then use the molecular biology software Bioedit to perform homologous comparison analysis on the downloaded sequences to find Highly homologous conserved sequences were used as candidate regions for primer and probe design, and primer design was performed in conjunction with the software PrimerSelect. The core design idea is: comprehensively consider the specificity and versatility of the detection of diarrhea virus (use degenerate bases at the viral nucleic acid mutation sites), and the universal principles that should be followed in primer design (such as Tm value, 3' terminal free energy , GC content, avoidance of internal secondary structure and dimer formation, etc.), and the interaction between primers during multiple amplification reactions, etc. After the desig...

Embodiment 2

[0049] Embodiment 2: The composition and detection method of the kit for detecting four kinds of diarrhea viruses based on HRMA technology

[0050] 1. The composition of the kit (stored at -20°C)

[0051] (1) 2X amplification reaction solution: its components are: 100mM Tris-HCl (pH8.3), 100mM KCl, 20mM MgCl2, 20mM DTT, 1mM Spermidine, 0.1% Tween-20, 0.2mg / L BSA, 0.4mM dNTP, 2.4 μM EvaGreen, 1.2 μM primer F1, 1.2 μM primer R1, 0.4 μM primer F2, 0.4 μM primer R2, 1.6 μM primer F3, 1.6 μM primer R3, 1.6 μM primer F4, 1.6 μM primer R4; wherein primer F1 is The sequence number is the nucleotide sequence shown in SEQ ID NO: 1, the primer R1 is the nucleotide sequence shown in SEQ ID NO: 2, and the primer F2 is the nucleic acid sequence shown in SEQ ID NO: 3. Nucleotide sequence, primer R2 is the nucleotide sequence shown in SEQ ID NO: 4, primer F3 is the nucleotide sequence shown in SEQ ID NO: 5, primer R3 is the sequence number shown in SEQ ID The nucleotide sequence shown in NO...

Embodiment 3

[0064] Example 3: Sensitivity analysis of the kit for detecting four diarrhea viruses based on HRMA technology

[0065] 1. Method:

[0066] (1) Sample processing: Use the above-mentioned viral RNA transcribed in vitro as a template for detection, use a micro-ultraviolet spectrophotometer to measure the concentration of the purified RNA, calculate the copy number of the initial RNA template by molecular weight, and then make it 10 times in turn Gradiently diluted to single copy number, a total of 9 gradients, the copy number is: 1×10 8 to 1×10 0 .

[0067] (2) Parallel control experiment: respectively select the HRMA detection kit of the present invention and common RT-PCR technology (basic reagents are purchased from Treasure Bioengineering (Dalian) Co., Ltd., article number DRR055A) to the above-mentioned 9 gradient dilution samples and 1 Negative controls were used for amplification testing.

[0068] HRMA detection: The detection method described in Example 2 was used to...

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Abstract

The invention discloses a primer group which is based on a high-resolution melting curve analysis technique and used for simultaneously detecting four diarrhea viruses, and a quadruple detection kit containing primers. The kit is capable of specifically detecting four diarrhea viruses, that is, rotavirus, astrovirus, norovirus and sapovirus. Through comparative analysis on respective homologies of the four viral genomes, respective conserved sequence design primers with high homologies can be found, the four diarrhea viruses can be simultaneously detected by using the high-resolution melting curve analysis technique, the primer group has the characteristics of being simple, convenient, rapid, cheap, safe, high in sensitivity and high in specificity, and powerful technical support can be provided for prevention and control and clinical diagnosis of dysentery.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a rapid detection kit for diarrhea virus based on high resolution melting curve analysis technology (High Resolution Melting Analysis, HRMA), and the four kinds of diarrhea virus (rotavirus) used in the kit Virus, Astrovirus, Norovirus, Saruvirus) have four pairs of specific primers. Background technique [0002] Diarrhea disease is an important public health problem that seriously affects human health, especially in developing countries such as Asia, Africa and Latin America. Statistics from the World Health Organization (WHO) show that 1 billion infants and young children under the age of 5 worldwide suffer from diarrhea every year, and 5 million of them die. In China, diarrheal disease is the second most common disease after respiratory diseases, and it is also one of the two major causes of infant mortality. Viral diarrhea accounts for an important proportion of diarrhea...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2600/166
Inventor 汪海波谭华冯子力伍碧梅李艳兰赵俊华王琪莫秋华杨泽林继灿
Owner 珠海国际旅行卫生保健中心
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