Establishment Of RT-PCR And Real-time RT-PCR Method Detection Of Chicken Stunt And Immunosupression Syndome Virus

Chicken Stunt and Immunosuppression Syndrome Virus (tentative name, abbre- viation:CSISV) was an importance infectious virus that mainly causes the suppression of growth and immunity on poultry. Based on the prophase research,carried out the d- etection method of regular and quantitative RT-PCR.1.According to the completed sequences(loacated 23749-24288)of CSISV, a pair of specific primers F1,F2 was designed and synthesized to amplify a 540bp nucleotide fragment, consistent with the expected fragment size.Through the primer,dNTP and the reactive condition were optimized.The optical content of primer,dNTP was 40μmol/L, 2.5pmol/μL,the annealing temperature was 57℃and 35 cycle.The RT-PCR method for detection of CSISV was established.The specificity test indicated that the pair of primer could not amplify NDV,IBV-G strain,IBV-HN strain,IBDV.The reproducibility and sensitivi- ty test indicated that the method had strong repeatability,capable of detecting the virus content of 1.0856ng/μL,using had been established RT-PCR detedtion method of CSIS- V to repeated five times had the same results.Compared with conventional serological methods,RT-PCR is more rapidly performed,accurate,sensitive and more effectively.21 artificial conteracting toxic(9-120h) substances specimens(kidney,trachea,heart) were detected by using established RT-PCR repeated three times,and 20 of them were positi- ve.one of them was negative.2.The specific primer(F3,F4 ) and probe were designed according to the complet- ed completed sequences(located 26085-26180)of CSISV,Through the probe,primer, dNTP and the reactive condition were optimized.TaqMann RT-PCR was established by series of experiments including the selection and optimization of count of probes, primers, dNTP , Mgcl2 and annealing temperature. The optical content of primer,dNT -P, primer was10μmol/L, 10μmol/L ,25μmol/L /μL,the annealing temperature was 57℃and 35 cycle. The specificity test indicated that no any cross-reaction with NDV,IBV-G strain,IBV-HNstrain,IBDV;The RT-PCR assay could detect 7.5×1010~4.8×106 copy/μL of reversed cDNA.while the sensitivity of routine RT-PCR was 2.4×107copy/μL,The repeated test showed that the intra-assay and interassay were 0.2703% and 0.9079% respectively.These results showed that this method was sensitive,specific and fast. From the extraction of nucleic acid to reverse transcription to TaqMan RQ RT-PCR the hole process only needed 2h.Compare with regular RT-PCR more faster,more sensitive.21 a- rtificial conteracting toxic(9~120h) substances specimens(kidney,trachea,heart) were detectedby using established TaqMan RQ RT-PCR method repeated three times,all sam- ple were positive.