Sequence Of Endogenous ALV And Development And Application Of Real-time PCR For Detection Of Avian Leukosis Virus Subgroup J

The corresponding primers were designed to amplify a strain of endogenous E from commercial layers. The sequence of ALV-E expansion from commercial layers was homology of99.1%comparied with ev-1, and was homology of98.9%compairied with SD0501. Besides, it was homology of85.4%comparied with HPRS103and NX0101. And it was only homology of54.8%comparied with ev-3.This article studied the organs distribution of avian leukosis virus subgroup J (ALV-J) in birds, and established the real-time PCR. A SYBR Green I-based real-time PCR was developed for detection of ALV-J by using a pair of primers which was designed to target NX0101(5257-5510bp). The chicken β-actin gene was used as an internal control.The PCR product of ALV-J (253bp) and β-actin (139bp) were respectively linked to pMD18-T vector served as standard curve, and respectively named them PMD-18-J and PMD-18-β. The method of sensitivity, specificity and repeatability was conducted research. The results showed a precise linear relationship with a correlation coefficient of R2=0.9964; the detection limits was100copies of DNA plasmid, it was100times more sensitive than RT-PCR. The melting curve showed a single peak could only be detected for ALV-J. The variation coefficient of repeatability tests were less than5%. The developed real-time PCR assay could quickly detect ALV-J gene in expansion range with high efficiency, thus providing the basis for the organs distribution of avian leukosis virus subgroup J (ALV-J) in birds.The heart, liver, spleen, lung, kidney, thymus, bursa of fabric us and gland stomach of SPF chicken which were infected after8weeks were tested by real-time PCR. Their RNAs were respectively extracted and reverse transcriped to CDNAs. The gene copies of J and β-actin were calculated according to standard curve, and then the relative expression of ALV-J was caculated according to formula:relative expression of ALV-J=copies of ALV-J/β-actin. The results illustrated that relative expression of ALV-J in bursa of fabric us was highest, spleen and thymus was second, liver and gland stomach was third, but no difference between them. Relative expression in heart, lung and kidney was lowest. The sick chicken which their livers exsited tumors. Each organ was detected by real-time PCR. The datas showed that the relative ALV-J gene expression was highest, the thymus, bursa of fabric us and spleen were second, but no differences among them. Kidney, lung, and glandular stomach were in third. There were also no differences among them. And Heart was the lowest.ALV-J gene distribution of organs in chicken was studied by using SYBR Green I real-time PCR. This showed that the total ALV-J relative content about clinically infected chicken was significantly higher than artificially infected chicken.