Establishment And Application Of A Multiplex PCR To Determine PRRSV,PCV2 And CSFV

The infectious diseases caused by porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus 2 type(PCV2) and classical swine fever virus(CSFV) are becoming the main threat to the development of swine-breeding industry,and leading to tremendous economic losses annually.The three infectious diseases induce reproduction barrier and disease of respiratory system to pigs and The extreme similarities of the three infectious diseases in their clinical manifestations make it more difficult to diagnose.However,the correct diagnosis is critical prior to the effective treatment.At present,the routine diagnosis of these diseases are pathogen isolated and serum technology,etal.But,the former is trouble,time consumed and lowly detective rate.and the latter exists the default of non-specific reaction and can not distinguish between natural infection and immunization positive though it is simple and quick.Some scholars have established a PCR for testing PRRSV,PCV2 and CSFV,but the multiplex PCR for the three viruses is none.Aimed at the early and specific identification of the relevant infection,a multiplex PCR has been designed and tested in the present study.1 Establishment of multiplex PCR for PRRSV,PCV2 and CSFVFirst,three pairs of specific primers were separately designed according to the analysis of the highly conservative DNA sequences within ORF7 of PRRSV American strain(ATCCVR-2332),ORF2 of PCV2(AF381175) strain,and E2 gene of CSFV.The multiplex PCR-working conditions were then optimized in terms of magnesium ion concentration,the combination of primer concentrations,and the annealing temperatures.After a series of trials,we finally established the effective multiplex PCR methodology for the detection of PRRSV,PCV2 and CSFV.It demonstrated that this method is characterized by rapidity,specificity,as well as sensitivity.Using this PCR system,the minimum detectable amounts of DNA for PRRSV,PCV2,and CSFV are respectively 4.8pg,5.0pg,and 14.5pg.2 The clinical application of the multiplex PCR for the detection of PRRSV,PCV2 and CSFVOne hundred and twenty samples(including lymphocytes,lungs,livers,spleens and kidneys) from sick pigs were collected from the below ten areas within Anhui province:Hefei,Anqing,Liuan,Bengbu,Chuzhou,Chaohu,Huaibei,Suzhou,Fuyang and Chizhou.The multiplex PCR was then conducted in order to determine the pathogenic viruses.The positive ratio of PRRSV,PCV2 and CSFV was 58.33%,49.17%and 23.33%separately,except that PRRSV and CSFV were isolated from Fuyang area.With the statistical analysis of biological,the infections of PRRSV and PCV2 was the most serious and both appeared an increasing trend in Anhui Province.Moreover,the PRRSV infection distributed in a regionally different fashion, since the infection occurred in Anqing,Liuan,and Chuzhou were more serious than in other areas.The co-infection among PRRSV,PCV2 and CSFV was detected,The results were that,the positive ratio of,PRRSV and CSFV,PCV2 and CSFV,three of PRRSV,PCV2,CSFV was respectively 32.50%,17.50%,9.17%,6.67%.With the statistical analysis of biological,the co-infection among PRRSV,PCV2 and CSFV were prevalent,in which the PCV2 and PRRSV dual infections accounted for the majority.3 The comparison between HPPRRSV(high pathogenic porcine reproductive and respiratory syndrome virus)(Nsp2 1594～1680 variation)RT-PCR test reagent kit and the PCR of PRRSVSixty-five samples collected from sick pigs in eight different areas(Hefei, Anqing,Liuan,Chuzhou,Chaohu,Huaibei,Suzhou,and Chizhou) in Anhui province were tested in parallel with HPPRRSV RT-PCR kit(way 1) and our PCR method(way 2).The positive ratio by way 1 was 73.84%,and way 2 was 75.38%.The coincidence rate of two methods peaked as 86.15%.With the statistical analysis of biological,at present,the variant infections of PRRSV is overwhelming in Anhui province.Above all,the established multiplex PCR assay is quick,specific,sensitive and has important clinical significance.