Recombinase-aid amplification (RAA) thermostatic fluorescence detection method of infectious hypodermal and hematopoietic necrosis virus (IHHNV), and reagent

Abstract

The invention discloses a recombinase-aid amplification (RAA) thermostatic fluorescence detection method of infectious hypodermal and hematopoietic necrosis virus (IHHNV) of prawns, and a detection kit. The detection kit comprises a forward primer shown in SEQ ID No. 1, a reverse primer shown in SEQ ID No. 2, a specific fluorescent probe shown in SEQ ID No. 3, a reaction solution, recombinant polymerase and a reference substance. The kit provided by the invention is high in specificity; the detection sensitivity is high, and can reach up to 0.32fg/mu L; the detection method has the advantagesof high accuracy, reliability, simple, convenient and fast operation, and suitability for field detection, thus having wide application prospect.

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, the pathogen of DRS). The disease was first discovered in a farm in Hawaii, USA in 1981. It first broke out in Litopenaeus stylirostris (L. stylirostris). Subsequent investigations found that it also widely exists in shrimp cultured in Asia and America. This disease is particularly serious to the harm of juvenile shrimp, and it affects the development of whole prawn farming industry. Epidemic outbrea...

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

Images