Construction and Preliminary Application of RT-LAMP Detection Method for Swine Japanese B Encephalitis Virus

[ Objective] This study aimed to establish a rapid, sensitive and specific method using reverse transcription loop-mediated isothermal amplification （RT-LAMP） technology to detect swine Japanese B encephalitis virus （JEV）. [ Method ] Four specific LAMP primers were designed according to six loci the conservative region of JEV E gene sequence. Positive JEV RNA sample was used as a template for one-step amplification, and the reaction conditions and reaction system were optimized. [ Result] Experimental results showed that the established method had high sensitivity, with the detection limit of 0.5pg; specificity experi- ments indicated that the method had high specificity and there was no amplification reaction for other viral pathogens. The coincidence rate between detection results of RT-LAMP and RT-Nested-PCR was 90.9%. After RT-LAMP reaction, a chemiluminescent agent was added for visual observation, which greatly reduced the detection time. This method required no special equipment but only a water bath, which was a simple, sensitive and rapid detection method for swine Japanese B encephalitis virus and could be applied in primary laboratories. [ Conclusion] An RT-LAMP detection method for swine Japanese B encephalitis virus was successfully established and preliminarily applied in clinical practice.

Establishment on Genotype Identification PCR Detection of Porcine Circovirus Type 2

ORF2 gene fragment was amplified from Porcine circovirus type 2（ PCV2） Shandong isolates by PCR,homology analysis and genotype identification were conducted on PCV2 ORF2 gene combining with the sequences listed in Gen Bank. The dominating popular genotype of PCV2 in Shandong Province was developed and the gene genetic variation characteristics of PCV2 was discussed. Specific PCR primers were designed and synthesized according to the differences among PCV2 genetypes,PCV2 gene identification PCR detection was established,and feasibility of the method was evaluated from the respects of sensitivity,specificity and repeatability,etc A total of 28 PCV2 ORF2 complete genes were obtained including 4 PCV2 a isolates,21 PCV2 b isolates and 3 PCV2 d isolates,indicating that the dominating popular genotype of PCV2 in Shandong Province was PCV2 b. Analysis on gene homology showed that homology of isolates in different regions from2000 to 2012 was 90. 5%- 99. 8%,amino acid sites sequences existed in different genotypes of PCV2 were different. Fragments amplified by the established PCR method were 341 bp and 177 bp,the lowest content of DNA could be detected was 5 ng / μL. Specific test results showed that the DNA amplifications on PCV2 a and PCV2 b were both positive,while amplifications on Porcine circovirus virus 1（ PCV1）,Porcine reproductive and respiratory syndrome virus（ PRRSV）,Pseudorabies virus（ PRV）,Porcine parvovirus（ PPV）,Classical swine fever virus（ CSFV）,Japanese encephalitis virus（ JEV） and Escherichia coli（ E. coli） were all negative,all the tests were conducted for 10 times,and the results were consistent. Test results indicated that the established PCR method had good specificity and repeatability,which could be applied to the identification of PCV2 genotypes.