Development And Application Of SYBRGreen â…  Fluorescent Quantitative RT-PCR Assay For The Detection Of Subgroup J Avian Leukosis Virus

Subgroup J Avian Leukosis referred to chicken myeloid cell tumor and othertumor diseases caused by Avian Leukosis Virus subgroup J (ALV-J). ALV-J wasisolated from commercial broilers for the first time in1999in China. ALV-J hasspread widely over China since2000, and its host range extended from meat-typechickens to laying hens and local chicken breeds. Production ability of infectedchickens was decreased and the morality was quickly increasing, which had broughtgreat losses for domestic poultry industry. Therefore, the establishment of a rapid,specific and sensitive method for detecting ALV-J is of great significance.In this study, a SYBR GreenⅠ-based fluorescent quantitative PCR assay fordetecting Avian Leukosis Virus subgroup J （ALV-J）was developed and applied. Theconservative region between pol and gp85gene of the HPRS-103strain of ALV-J wasamplified by conventional PCR and was cloned into pMD18-T vector served aspositive standard to establish fluorescent quantitative PCR standard curve. And thenthe detecting method of ALV-J SYBR Green I fluorescent quantitative PCR wasrealized by optimizing template concentration, primer concentration and annealingtemperature. The results indicated that the method was sensitive with a detection limitof2360copies/μL of positive recombinant plasmid, which was100time moresensitive than the conventional PCR, and was highly specific without cross-reaction toother avian viruses. The coefficient of variation for intra-and inter-assay was bothless than5%. It indicated that this method was of high specificity, good repeatedstability and sensitivity.125clinical suspected ALV-J disease chickens from four different varieties andcommercial poultry vaccines were detected by the assay established and theconventional method respectively, the ALV-J positive rate was46.40%(58/125), ofwhich yellow broiler chicken was65.31%(32/49), huainan spotted-brown chickenwas55.26%(21/38), local chicken breeds was31.25%(5/16) and Jinghong NO.1laying hens was0%（0/22）, respectively. It indicated that different varieties ofchickens had distinct susceptibility. No ALV-J was detected in12different poultryvaccines, which indicated that there was no ALV-J pollution in detected poultryattenuated vaccines sold in the market.Moreover, the assay was used to detect the transcripts levels of ALV-J gene in theorgan samples, including heart, liver, spleen, lung and kidney from ten positive yellowbroiler chicken of27weeks. As a result, virus genes were to found in all organs detected and the kidney had the highest viral gene load than the other organs bymultiple comparative tests, and no significant difference existed among the lungs,heart, liver, and spleen. It laid a foundation for further study of the interactionbetween the virus and the organs, and its pathogenesis.In a summary, fluorescent quantitative RT-PCR method established in the studyis a quick (about6h), specific and sensitive testing method, which could be applied todetect clinical suspicious sample, eradication and the distribution of virus in differentorgans and its expression.