Raa constant temperature fluorescence detection method and reagents for infectious hypodermic and hematopoietic necrosis virus (IHHNV)

Abstract

The invention discloses a constant-temperature fluorescence detection method and a detection kit for shrimp infectious subcutaneous and hematopoietic necrosis virus (IHHNV) RAA. The detection kit includes a forward primer of SEQ ID NO.1, a reverse primer of SEQ ID NO.2, a specific fluorescent probe of SEQ ID NO.3, a reaction solution, a recombinant polymerase and a control substance. The kit of the invention has strong specificity; high detection sensitivity, which can reach 0.32fg / μL; high accuracy and reliability; simple and fast operation, suitable for on-site detection, and has wide application scenarios.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method of marine aquaculture industry, in particular to a RAA constant-temperature fluorescence detection method and a kit for infectious subcutaneous and hematopoietic tissue necrosis virus of prawns. Background technique [0002] Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is the source of disease causing shrimp infectious hypodermal and hematopoietic necrosis (IHHN), that is, causing chronic short stature syndrome ( Runt-deformity syndrome, DRS) pathogen. The disease was first discovered in 1981 in a farm in the Hawaii region of the United States, and the first outbreak occurred in Litopenaeus stylirostris ( L. stylirostris ), subsequent investigations found that it was also widely present in cultured shrimp in Asia and America. This disease is particularly serious to the harm of juvenile shrimp, and it affects the development of whole ...

AI Technical Summary

Problems solved by technology

Although electron microscope counting can directly observe the presence of virus particles, the operation is complicated, time-consuming and costly

The HE staining method is based on pathological counts, and cannot directly detect the virus, but can only be detected by using the histopathological signs of the disease

Antibody detection method and nucleic acid probe hybridization method are used to detect the protein or nucleic acid components of the virus itself. The detection sensitivity is low and time-consuming, and can only be used for the detection of diseased shrimp or shrimps that are about to become diseased; IHHNV has not yet caused infection or in Infection is very early and difficult to detect with the above methods

Although the PCR detection method of IHHNV overcomes the shortcomings of the first four methods, it can achieve relatively fast and accurate detection of IHHNV under laboratory conditions, but because conventional PCR detection requires expensive PCR equipment, gel electrophoresis and imaging systems, And the detection time is long, which also greatly limits the popularization and application of PCR detection method in practice.

LAMP isothermal amplification technology also has high false positives and low accuracy, so its application in the detection of aquatic pathogens is still relatively limited

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