The Establishment And Preliminary Application Of A Multiplex PCR To Determine PRRSV, PCV2and SS2

The infection diseases caused by porcine reproductive and respiratory syndromevirus(PRRSV), porcine circovirus2type(PCV2) and Streptococcus suis2type are becomingthe main threat to the development of swine-breeding industry, and leading to tremendouseconomic losses annually. The three infections diseases induce disease of respiratorysystem to pigs and the extreme similarities of the three infectious diseases in their clinicalmanifestations make it more difficult to diagnose.However, the correct diagnosis is ctiticalprior to the effective treatment.First, three pairs of specific were separately designed according to the analysis of thehighly conservative DNA sequences within nsp2of PRRSV, whole-genome of PCV2and16s rRNA of SS2.The multiplex PCR-working conditions were then optimized in terms ofmagnesium ion concentration, the combination of primer concentrations,and the annealingtemperatures.Theoptimal annealing temperature was52℃, the optimalnumber of cycleswas40.The reaction conditions singlet PCR method for detection of the three pathogenswere identified as the annealing temperature is51.9℃, the results of a selected number ofcycles to35cycles; among PCV2and SS2singlet PCR reaction system (25μL System)dNTP (2.5mM) was added to determine the volume of3μL, primers (10μmol/L) wasadded to determine the volume of0.7μL; singlet PRRSV in the PCR reaction system (50μL system) Mg2+(25mM) was added to determine the amount of8μL, dNTP (10mM)was added to determine the amount of5μL, primers (10μmol/L) was added to determinethe volume of2.3μL. Using this single PCR system, the minimum detectable amounts ofRNA for PRRSV, DNA of PCV2, and SS2are respectively1.37pg,3.35pg, and5.23pg.The reaction conditions multiplex PCR method three pathogens in the annealingtemperature were determined to be51.9℃, Mg2+(25mM) added to the amountdetermined to be8μL, dNTP (10mM) added to the amount determined as6μL, primers(10μmol/L) was added to determine the volume of2.3μL, the results of the determinationof the number of cycles35cycles. Using this multiplex PCR system, the minimumdetectable amounts of RNA for PRRSV, DNA of PCV2, and SS2are respectively13.7pg,33.5pg, and52.3pg. Fore hundred and twelve samples (including lymphocytes, lungs, and spleens) fromsick pigs were collected from the below eleven areas within hebei province:baoding,Shijiazhuang, Handan, Xingtai, Cangzhou, Chengde, Zhangjiakou, Hengshui,Langfang, Tangshan, Qinhuangdao from September2012to December2013. The positiveratio of PRRSV, PCV2and SS2was42.7%,26.5%and44.7%. The co-infection amongPRRSV, PCV2and SS2were detected. The results were that, PCV2and PRRSV, PCV2SS2, PRRSV and SS2was15.0%,12.4%, and11.2%. The results showed that PRRSV,PCV2and SS2infections prevalent in areas in which herds of Hebei Province.