Quantitative detection kit and detection method for virus content of lapinized classical swine fever virus vaccine

Abstract

The invention provides a fluorescent quantitative (PCR) rapid diagnostic kit for quantitatively detecting virus content of spleen and lymph tissue origin and cell culture origin classical swine fever virus C-strain vaccine, and a preparation method thereof. The method can achieve specific amplification when a total RNA content of the virus is 10<2> copies/[mu]L, has a good amplification curve in a linear range of 10<7>-10<2> copies/[mu]L, is suitable for titration evaluation of finished products and half-finished products of the lapinized classical swine fever virus vaccine. The specific primers of the classical swine fever virus (CSFV) used by the invention is two primers designed and synthesized in highly conserved 5' untranslated region (5'-UTR), wherein P1(5'-GCAGAAGCCCACCTCGAGAT-3') is a specific upstream primer of the classical swine fever virus; P2(5'-TACACCGGTTCCTCCACTCC-3') is a specific downstream primer of the classical swine fever virus; an expected amplification product is 245 bp; fluorescent quantitative (PCR) reaction presents a characteristic S-type amplification dynamic curve; and analysis of a melting curve shows a single characteristic peak. The method can only detect CSFV, and has no cross-reaction with BVDV.

Description

technical field [0001] The invention relates to a method and a kit suitable for the quantitative detection of spleen-derived and cell-derived virus content of a classical swine fever rabbitized attenuated vaccine strain. It can be used for real-time monitoring and quality testing of the production of the attenuated hog vaccine. technical background [0002] Classical swine fever is an acute, highly contagious disease caused by classical swine fever virus (CSFV), with a mortality rate of over 90%. It is listed as an animal disease that must be notified by the World Organization for Animal Health (OIE). At present, the disease is mainly prevalent in Asia, Europe, and South America, and is widely spread in domestic pigs and wild boars. Developed countries have adopted hunting policies to eliminate swine fever, which has caused significant economic losses to the pig industry. [0003] CSFV has certain antigenic cross-reactivity with bovine viral diarrhea virus and sheep border ...

AI Technical Summary

Problems solved by technology

The level of vaccine titer is the key indicator of whether the vaccine is qualified or not. Although HCLV can grow on a variety of cells, the virus multiplication ability is relatively weak, and it does not cause cytopathic changes. The virus titer is low, and it is difficult to apply immunofluorescence technology. Detects the presence of antigens in infected cells

The effectiveness test of the rabbit-type attenuated vaccine against classical swine fever has been using the rabbit stereotyped heat reaction method, but the prominent shortcomings of this method are that it cannot be accurately quantified, the test results are easily affected by individual differences in rabbits and weather conditions, and the experiment cycle is long and time-consuming. Effortlessly wait

Images