RT-RPA kit, primer and probe for visually and rapidly detecting Chinese softshell turtle hemorrhagic syndrome virus TSHSV

A RT-RPA, syndrome virus technology, applied in the field of biological detection, can solve the problems of inability to judge samples, confusion of disease samples, inability to diagnose, etc., and achieve the effects of clear visual detection, specific detection results, and short detection time.

Pending Publication Date: 2022-03-15
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical diagnosis is only for diseased samples, and it cannot be diagnosed. It is easy to be confused with diseased samples with similar clinical symptoms, and it is even impossible to judge samples that carry the virus but do not have the disease; the nucleic acid detection methods established in the early stage are only applicable to those with professional equipment and professional Laboratory use for advanced technicians
At present, there is no rapid, visual and simple viral nucleic acid molecular detection method suitable for on-site detection of TSHSV

Method used

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  • RT-RPA kit, primer and probe for visually and rapidly detecting Chinese softshell turtle hemorrhagic syndrome virus TSHSV
  • RT-RPA kit, primer and probe for visually and rapidly detecting Chinese softshell turtle hemorrhagic syndrome virus TSHSV
  • RT-RPA kit, primer and probe for visually and rapidly detecting Chinese softshell turtle hemorrhagic syndrome virus TSHSV

Examples

Experimental program
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Effect test

Embodiment 1

[0081] An RT-RPA kit for visual and rapid detection of Chinese soft-shelled turtle hemorrhagic syndrome virus TSHSV, said kit comprising: a dry powder reaction tube containing RPA freeze-dried powder; said RPA freeze-dried powder includes recombinant enzyme, single-stranded DNA binding protein , DNA polymerase, auxiliary protein and M-MLV reverse transcriptase (both enzyme and protein are from Weifang Anpu Future Biotechnology Co., Ltd.);

[0082] Buffer A, the composition of the buffer A is: 6wt% polyethylene glycol, Tris-HCL 30mM, ddH 2 O margin;

[0083] Buffer B, the buffer B is a magnesium acetate solution with a concentration of 280mM;

[0084] Primer, the sequence of the primer is:

[0085] Upstream primer TSHSV-F2: 5'-AAGCTTCCAGCCTATTGGCCTCCATTTACC-3'

[0086] Downstream primer TSHSV-R1: [5'-biotin]AATTGTCTTTCCCAACATACTCACTTCCGTTG-3';

[0087] A colloidal gold probe, the colloidal gold probe is TSHSV-nfo-PB, and its sequence is [5'-6FAM]TCTTGTGCTGGATGTTGGCGAATTCTTG...

Embodiment 2

[0090] The difference between this embodiment and Example 1 is that the sequence of the primer is:

[0091] Upstream primer TSHSV-F4: 5'-TTGTTGAGCAAGACCACCAAGGACGACCCT-3'

[0092] Downstream primer TSHSV-R2: [5'-biotin]AATCTCAGAGGTCAGCTTCTCAATAGTTTTG-3';

[0093] Others are with embodiment 1.

Embodiment 3

[0095]An RT-RPA kit for visual and rapid detection of Chinese soft-shelled turtle hemorrhagic syndrome virus TSHSV, said kit comprising: a dry powder reaction tube containing RPA freeze-dried powder; said RPA freeze-dried powder includes recombinant enzyme, single-stranded DNA binding protein , DNA polymerase, auxiliary protein and M-MLV reverse transcriptase (both enzyme and protein are from Weifang Anpu Future Biotechnology Co., Ltd.);

[0096] Buffer A, the composition of the buffer A is: 6wt% polyethylene glycol, Tris-HCL 30mM, ddH 2 O margin;

[0097] Buffer B, the buffer B is a magnesium acetate solution with a concentration of 280mM;

[0098] Primer, the sequence of the primer is:

[0099] Upstream primer TSHSV-F2: 5'-AAGCTTCCAGCCTATTGGCCTCCATTTACC-3'

[0100] Downstream primer TSHSV-R1: [5'-biotin]AATTGTCTTTCCCAACATACTCACTTCCGTTG-3';

[0101] A fluorescent probe, the fluorescent probe being TSHSV-exo-PB,

[0102] Its sequence is: 5'-TCTTGGCCTTGAACCAGTATACTGTTACAAA...

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Abstract

The invention discloses an RT-RPA kit, a primer and a probe for visually and rapidly detecting the hemorrhagic syndrome virus TSHSV of Chinese softshell turtles. The kit comprises a dry powder reaction tube containing RPA freeze-dried powder; the kit comprises a buffer solution A, a buffer solution B, a primer, a colloidal gold probe and a colloidal gold test strip. The detection result is specific, the result can be judged by naked eyes, the detection time is short, the operation is simple, convenient and rapid, and the kit is suitable for definite diagnosis, screening and prevention of the Chinese softshell turtle hemorrhagic syndrome virus.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an RT-RPA kit, primers and probes for visual and rapid detection of Chinese soft-shelled turtle hemorrhagic syndrome virus TSHSV. Background technique [0002] The Chinese soft-shelled turtle (Trionyx sinensis) belongs to the class Reptilia in taxonomy, and is mainly distributed in China, Japan, Southeast Asia and other countries and regions. Because of its unique nutritional and health value, it has become an important freshwater aquaculture species in my country, mainly distributed in Zhejiang, Guangdong, Guangxi, Hunan, Hubei, Jiangxi and other provinces. Disease is an important limiting factor for the healthy and sustainable development of soft-shelled turtle aquaculture. Compared with fish diseases, basic research on soft-shelled turtle diseases is relatively weak, most of which are limited to clinical diagnosis and treatment trials of diseases, and less resear...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844G01N33/543C12N15/11
CPCC12Q1/701C12Q1/6844G01N33/54313C12Q2521/507C12Q2522/101C12Q2521/107
Inventor 刘莉吕孙建郭琦沈卫锋韩明明
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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