The Study And Application Of RT-PCR Kit For Infectious Bronchitis Virus Detection

In order to find appropriate RNA extraction method for IBV, three different RNA extraction methods (iso-sulfuric cyanic guanidine, Trizol reagent and RNA extraction kit) were compared and evaluated. UV and electrophoresis were employed to test the purity and integrity of extracted RNA. The result shows: The RNA which extracted by so-sulfuric cyanic guanidine has got the lowest purity and integrity. And the RNA which extracted by Trizol reagent and RNA extraction kit has got good purity and integrity. For the difference of extraction efficiency between RNA extraction kit and Trizol reagent is not significant, considering the cost, Trizol reagent is chosen for next steps of experiment.A pair of primers located at the conservative district of IBV nonstructural gene 3abc was designed, and the specificity of the primers was also tested; One-step RT-PCR was established and optimized, and then one-step RT-PCR kit was constructed according to the optimized condition and concentration. The result shows: when the primers were employed to detected IBV isolates and IBV standard strains, positive results were got; when it came to IBV DNA vaccine and other pathogens of chicken, negative results were got. The positive products were collected and sequenced, and the result shows: the positive product was 3abc gene of IBV.In order to test the specificity of the kit, 8 domestic IBV isolates, 3 domestic standard strains, IBV DNA vaccine (S1, M and N gene mixed) and 4 other pathogens of chicken were detected by the kit. The result shows: positive results were got when IBV isolates and IBV standard strains were detected, negative results were got when IBV DNA vaccine and other pathogens of chicken were detected. Different concentration of IBV RNA were dectected by the detection kit, the result shows: the concentration of 10^-3 ng/μl RNA template can be detected at least. In order to test the stability and reproducibility of the detection kit, IBV strains, IB DNA vaccine and other pathogens of chicken were detected regularly for 6 monthes since the construction of the kit. The result shows: the kit kept good stability and specificity.