Method for preparing positive control for single-tube RT-PCR detection of three fish disease virus

Abstract

The invention relates to a method of make single valve RT-PCR dectecting masculine comparing virulence haemorrhagic septicemia virus, fish infectivity hemocytopoietic organ putrescence and infectivity pancreas putrescence germina. Its feature is selecting G gene from virulence haemorrhagic septicemia virus, VP gene from fish infectivity hemocytopoietic organ putrescence and N gene from infectivity pancreas putrescence germina, inserting into pUC57 particle carrier and transfect to DH5 alpha coliform bacteria to take in vitro expression, and extending the section 625bp, 371bp, 206bp. It has very high use value.

Description

technical field [0001] The invention relates to a method for preparing a positive control for detecting aquatic animals, especially fish viruses, in particular to a single tube of viral hemorrhagic sepsis virus, fish infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus Preparation method of positive control for RT-PCR detection. Background technique [0002] Viral hemorrhagic septicemia virus (VHSV), fish infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are listed as the first and second class infectious diseases of imported animals in the People's Republic of China. VHS and IHN are the second-class and third-class infectious diseases stipulated in the "Animal Epidemic Prevention Law of the People's Republic of China" respectively. These three diseases are listed in the OIE list of the International Office of Epizootics. Both IHN and VHS viruses belong to the single-stranded RNA vi...

AI Technical Summary

Problems solved by technology

At present, the commonly used detection method for the three fish diseases is to inoculate sensitive fish and cells with disease materials for virus propagation, isolate and purify the virus, and then use serum neutralization, immunofluorescence and enzyme-linked immunoassay methods for virus identification. The operation steps are complicated and tedious , up to 2-4 weeks

Since these three viruses are all RNA viruses, using virus-infected materials to extract RNA for RT-PCR reactions requires high requirements for experimental consumables such as test tubes and pipettes, and the processing is more complicated to eliminate RNases, because RNA is susceptible to The environment is degraded by RNase. The extracted RNA positive control can only be stored for about 1 week by conventional methods. Direct use of virus-infected materials can easily cause virus spread, so it is inconvenient to be used as a positive control for commercial kits.

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