Study Of Real-time Quantitative PCR For Detection Of Exogenous ALV In Split Influenza Virus Vaccine

Influenza virus vaccine as an important means of prevention of the pandemic outbreak can effectively prevent flu-related illness and death. Now there is not specific medicine to cure anyone from getting the flu, but vaccination is regarded as the best method of influenza prevention. Fertilized hens' egg is the common choice in manufacturing influenza vaccine. In the 2010 edition of the Chinese Pharmacopoeia added influenza virus vaccine, which require using the enzyme-linked immunosorbent assay test exogenous avian leucosis virus in the master seed lot, test results should be negative.ALV detection is mainly reliant on virus isolation and ELISA. Serological diagnosis waste long time needing to prepare specific antibodies and exist Biological safety hazard. The development of molecular detection offers the possibility of early diagnosis. Application of ordinary RT-PCR method can be performed within 1 day of ALV. But the method is easy to cause the cross contamination between specimens and result to false-positive results. Most recently, real-time PCR has been developed to detect ALV. Real-time PCR technique, compared with conventional PCR technology, has higher sensitivity, specificity and reproducibility, and also has rapid, high throughput, low pollution characteristics.Completed sequences of ALV-A, ALV-B, ALV-C and ALV-J download from GenBank. Looking for is located in the LTR region of conserved sequence, and designed primers and probe. We diluted the standard plasmid which contained the conserved sequences 10 times serially. Appropriate amplification curve and standard curve obtained by fluorescence quantitative PCR. We got the correlation coefficient of the standard curve was R2=0.998, amplification efficiency was Eff=96.5%. So the standard curve has a good linear relationship.The standard plasmid was diluted from 109 to 101 copies/ ? L to compare the sensitivity. The sensitivity of the plasmid can reach 10-1copies/?L.The detection of exogenous ALV was positive, but the detections of the other Mouse leukosis virus, H1N1 influenza viruses and H7N9 influenza viruses were negative.We succeed to establish a method that can be used to detect the exogenous ALV. The real time PCR may realize to the fast detection of the exogenous ALV. This method has the advantages of fast, sensitive, specific and can quantitate the vaccine samples.