Chicken Gland Gastirtis Pathogen Isolation And Real-itme PCR For Deteciton Of Expression Of IBV In The Chicken Proventriculitis

The incidence of proventriculitis of broiler and laying hens during2011-2012was veryhigh. This disease had been found in most of the provinces in China, causing seriouseconomic losses to the poultry industry. It was of great significance to strengthen research ondetection of the disease for protecting and promoting the healthy development of the poultryindustry. Since the occurrence of proventriculitis, the etiology remained controversial.Infectious bronchitis virus was isolated in clinical cases of infectious and virusproventriculitis in this study. The sick chickens pathological changed according to differentdisease severity. Figuring out viruses target organ for the viral disease of unknown etiologyplayed a vital role in prevention and treatment of the disease. The real-time PCR was appliedto measure the content of the virus in sick chicken’s thymus, lungs, heart, proventriculus,kidney, liver, brain and other organs to do quantitative analysis and analysis of thesignificance of difference.Eight generations of allantoic fluid was passed consecutive blindly. The allantoic fluiddid not have hemagglutination. By NDV interference experiments and RTPCR experiments,we preliminarily identified that infectious bronchitis virus was pathogen. A pair of primerswere designed for IBV-N gene sequence and the RT-PCR reaction amplified target fragmentof216bp. Taking into the past published literature, primer of β-actin was compoundedampiying reference gene. The size was139bp. The two target amplification fragments wererestructuring into pEASY-T1Cloning Vector. The recombinant plasmids were namedpEASY-T1-N and pEASY-T1-β-actin. After two recombinant plasmid restriction enzyme andsequencing successfuly, the two recombinant plasmid were seened as a standard for theestablishment of quantitative PCR standard curve. The experiment results showed that: thelinear relationship R2value of standard curve was0.9972and0.9983; The correlation wasexcellent and the detection limit was1.0E+02copies.Various organs of proventriculitis cases were conducted RNA extraction. cDNA was thetemplated for quantitative PCR. The measured Ct values were substituted into the standard curve formula to calculate the copy number of N gene and β-actin gene. Then, according tothe formula: IBV-N relative expression amount=copy number of IBV-N/copy number ofβ-actincalculated the relative content of the IBV-N. Clinical samples were detected byestablished methods. The results showed that the highest relative content of all organs wasproventriculus followed by kidney. The results of Duncan-test showed that the comparision ofkidney and stomach P<0.01, the difference was very significant, we conducted t-test betweenkidneys and lungs, lungs and liver, liver and heart, heart and brain, brain and thymus. Theresults revealed that P>0.05and the difference was not signicant. Therefore we can concludedthat For the clinical cases manifestation of gastritis, the target organ was glandular stomachwhen infectious bronchitis virus infected.