Method for detecting fish contagious hemopoietic organ necrosis virus based on RPA

A technique for hematopoietic organ necrosis and infectivity, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of dependence, long reaction time, and limited application, and achieve strong specificity, The effect of high sensitivity

Inactive Publication Date: 2019-03-19
梁君妮
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Huge economic loss to the salmon farming industry
At present, IHNV detection methods mainly include cell culture separation method, immunological method (neutralization test method, indirect fluorescent antibody method and ELISA method) and molecular biology method (PCR method, PCR probe and LAMP method), while PCR method, PCR method The probe method is the most commonly used detection technology, but this technology basically relies on thermal cycle instruments with accurate temperature control, which limits its application in on-site detection
Although the LAMP method does not require a thermal cycler, it needs to design multiple pairs of primers, and the reaction time is longer, generally 60min to 90min

Method used

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  • Method for detecting fish contagious hemopoietic organ necrosis virus based on RPA
  • Method for detecting fish contagious hemopoietic organ necrosis virus based on RPA
  • Method for detecting fish contagious hemopoietic organ necrosis virus based on RPA

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Five viruses, IHNV, SVCV, EHNV, VHSV, and IPVN, were detected using the established RPA detection system. The electrophoresis results are shown in figure 1 . The results showed that the target band of 285bp could be detected in the IHNV virus sample, and the target band was not detected in other SVCV, EHNV, VHSV, and IPVN viruses, which proved that the method had good specificity.

Embodiment 2

[0033] The IHNV virus cDNA of measured concentration is carried out 10 times serial dilutions, obtains 10 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Six dilutions of different dilutions were tested with the established RPA detection system to investigate the sensitivity of the method. The results show that see figure 2 , 100, 10-1, 10-2, 10-3 dilutions can detect a specific band of 285bp, 10-4, 10-5 dilutions did not find the target fragment, the sensitivity is good, and the detection limit is 1.56ng / mL.

Embodiment 3

[0035] The 50 samples kept in the laboratory were tested by the established RPA method and common PCR method, see Table 1. As can be seen from the table, the RPA method has seven samples to detect positive, and the positive detection rate is 14.00%; the common PCR method has six samples to detect positive (one of which is a weak positive), and the positive detection rate is 12.00% %. The positive detection rate of the RPA method was slightly higher than that of the common PCR method, indicating that the RPA had higher sensitivity than the common PCR method.

[0036]

[0037]

[0038]

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Abstract

The invention relates to a method for detecting fish contagious hemopoietic organ necrosis virus based on RPA and belongs to the technical field of biodetection. The method comprises the following steps: determining RPA primers according to a virus G protein conserved sequence, an upstream primer: 5'-GATGAGTGGGAGGGCTTTCACGGATTGC-3' and a downstream primer: 5'-GCACGCGGCAACAGCAAGGAGGAGAACAAGG-3'; extracting virus RNA from an RNA extraction kit box, preparing cDNA from virus RNA through an inverse transcription kit and freezing the cDNA for later use; detecting impurity and content of cDNA undera nucleic acid protein analyzer according to OD260/OD280 and concentration value; preparing an RPA reaction system; and identifying an RPA product. The method has the beneficial effects that the specific primers are designed according to a gene G of an IHNV conserved sequence, and the detection method is simpler and more efficient, high in specificity and high in sensitivity, is not dependent on ahigh end instrument, is suitable for field rapid detection, and can control burst of fish epidemic diseases.

Description

technical field [0001] The invention relates to a method for detecting fish infectious hematopoietic organ necrosis virus based on RPA, which belongs to the technical field of biological detection. Background technique [0002] Infectious hematopoietic necrosis (IHN) is a major infection caused by the infectious hematopoietic necrosis virus (IHNV) of the Rhabdoviridae family Nora rhabdovirus (Novirhabdovirus) Serious infectious disease of salmon and trout. The disease mainly harms the fry of salmon trout and juveniles of the year, often causing 70% to 90% mortality of fry, and even up to 100% in some cases. Huge economic losses are caused to the salmon farming industry. At present, IHNV detection methods mainly include cell culture separation method, immunological method (neutralization test method, indirect fluorescent antibody method and ELISA method) and molecular biology method (PCR method, PCR probe and LAMP method), while PCR method, PCR method The probe method is t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2531/119
Inventor 梁君妮尹伟力段晓辉刘宁
Owner 梁君妮
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