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Specific primers, probes and rapid detection kits for detecting Chinese soft-shelled turtle parvovirus

A detection kit and parvovirus technology are applied in the field of rapid detection of target DNA fragments, which can solve the problems of death of Chinese soft-shelled turtle and hinder disease prevention work, and achieve the effects of high yield, overcoming long detection time and simple instrument.

Active Publication Date: 2019-05-31
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus was isolated from the Chinese soft-shelled turtle suffering from hepatitis and enteritis in 2012. It caused a large number of deaths in the breeding period of the Chinese soft-shelled turtle. immeasurable
This virus has not been reported at home and abroad. Since there is no detection kit and detection method for this virus so far, it has seriously hindered the prevention of diseases caused by this virus. Therefore, the development of this virus detection kit and the research of detection technology are particularly important.

Method used

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  • Specific primers, probes and rapid detection kits for detecting Chinese soft-shelled turtle parvovirus
  • Specific primers, probes and rapid detection kits for detecting Chinese soft-shelled turtle parvovirus
  • Specific primers, probes and rapid detection kits for detecting Chinese soft-shelled turtle parvovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] A specific primer set A for detecting Chinese soft-shelled turtle parvovirus, the primer set includes primer TSDPV-F3, primer TSDPV-B3, primer TSDPV-FIP and primer TSDPV-BIP,

[0055] The nucleotide sequence of the primer TSDPV-F3 is shown in SEQ ID NO: 1;

[0056] The nucleotide sequence of the primer TSDPV-B3 is shown in SEQ ID NO: 2;

[0057] The nucleotide sequence of the primer TSDPV-FIP is shown in SEQ ID NO: 3;

[0058] The nucleotide sequence of the primer TSDPV-BIP is shown in SEQ ID NO:4.

[0059] The primer-probe mixture for detecting Chinese soft-shelled turtle parvovirus consists of primer set B and Taqman fluorescent probe C. The 5' end of probe C is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0060] The primer set B comprises primer TSDPV-q165F and primer TSDPV-q165R;

[0061] The sequence of the primer TSDPV-q165F is shown in SEQ ID NO: 5;

[0062] The nucleotide sequence of C of the probe...

Embodiment 2

[0069] Chinese soft-shelled turtle parvovirus chain displacement isothermal amplification visualization rapid detection kit, including viral DNA extraction reagents and reaction reagents.

[0070] The viral DNA extraction reagent comprises the following components: lysate A with pH 8.0 whose components are 100mM Tris, 50mM EDTA, 500mMNaCl, 2% SDS, and 10mM β-mercaptoethanol; potassium acetate aqueous solution, concentration 5M; absolute ethanol ; DEPC water configuration containing 75% ethanol washing solution A; DEPC water.

[0071] Described reaction reagent, it comprises following component:

[0072] 1) Pre-reaction solution: 20mM Tris-HC1 with a pH of 8.8, 8mM magnesium sulfate, 14mM potassium chloride, 10mM sodium sulfate, 0.12% Tween-20, 1.4mM dNTP, 0.6M betaine, 0.15-0.25μM primer TSDPV-F3, 0.15-0.25 μM primer TSDPV-B3, 1.6-2.0 μM primer TSDPV-FIP and 1.6-2.0 μM primer TSDPV-BIP;

[0073] 2) Reaction enzyme: Bst DNA polymerase containing 8 active units per microliter;...

Embodiment 3

[0077] The application of the Chinese soft-shelled turtle parvovirus chain displacement isothermal amplification visualization rapid detection kit in the detection of Chinese soft-shelled turtle parvovirus includes the following steps:

[0078] 1) DNA extraction: take 30-80 mg of soft-shelled turtle liver or spleen tissue in a 1.5 mL centrifuge tube, grind and pulverize with a grinding rod, add lysate A in the DNA extraction reagent, mix well, keep warm at 65°C for 15 minutes, and Shake occasionally, add 150 μL of potassium acetate aqueous solution, ice-cool for 20 minutes, centrifuge at 15,000 rpm for 15 minutes at 4°C, transfer the supernatant to another centrifuge tube, add 0.7 times the volume of absolute ethanol, mix well, and precipitate on ice for 20 minutes, then 12,000 rpm Centrifuge for 10 minutes, discard the supernatant, wash the precipitate once with 75% ethanol, dry it, and add an appropriate amount of DEPC water to dissolve the DNA;

[0079] 2) Gene amplificatio...

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Abstract

The invention discloses specific primers, a probe and quick detection kits for detecting Chinese softshell turtle parvovirus. The Chinese softshell turtle parvovirus chain replacement isothermal amplification visual quick detection kit comprises a primer set A. The Chinese softshell turtle parvovirus fluorescent quantitative detection kit comprises a specific primer set B and a Taqman fluorescent probe C. By adopting the chain replacement enzyme, isothermal amplification and fluorescent quantitative technique, the capsid protein gene of the Chinese softshell turtle parvovirus is detected to detect the Chinese softshell turtle parvovirus. The invention develops the primer sets and kits for detecting Chinese softshell turtle parvovirus for the first time at home and abroad at present. The development of the kit lays the foundation for the monitoring and prevention of Chinese softshell turtle parvovirus disease.

Description

technical field [0001] The invention belongs to the field of rapid detection of target DNA fragments, and in particular relates to a specific primer, a probe and a rapid detection kit for detecting Chinese soft-shelled turtle parvovirus. Background technique [0002] Trionyx sinensis Dependoparvovirus (TSDPV) is a pathogenic virus that causes a large number of deaths in Chinese soft-shelled turtles. It often causes Chinese soft-shelled turtle hepatitis, intestinal catarrhal inflammation, and pancreatic punctate necrosis, which is extremely harmful to the Chinese soft-shelled turtle breeding industry. . TSDPV is a single-stranded DNA virus that belongs to the genus Dependent Parvovirus of the Parvoviridae Parvoviridae of the Order Parvoviridae. This virus is the second DNA virus that can infect soft-shelled turtles so far. The virus was isolated from the Chinese soft-shelled turtle suffering from hepatitis and enteritis in 2012. It caused a large number of deaths in the bree...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/6851C12Q1/701C12Q2531/119C12Q2563/107C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 潘晓艺袁雪梅沈锦玉蔺凌云姚嘉赟徐洋尹文林
Owner ZHEJIANG INST OF FRESH WATER FISHERIES