| Based on conserved sequences in ORF6 and ORF7 fragments of PRRSV standard American strain(ATCC VR-2332),and that in NP gene of SIV A/ Swine/ HeNan/ 703/ 2001 (H3N2) strain,two pairs of specific primers were respectively designed:(P1)5'AAG CCT CGT GTT GGG TG3'and(P2)5'TGA GGG TGA TGC TGT GAC3' for PRRSV; (SP1) 5'GAC CTT ATC TTT CTG GCT CG3'and(SP2) 5'ACA TC T TCT GGT CTG GCA CT3' for SIV.The total RNA were extracted respectively from PRRSV-infected Marc-145 cell culture and SIV-infected chicken embryo allantoic fluid and RT-PCRs were performed.The two fragments amplified,which were 426bp and 605bp respectively and were comsistent with target DNA.After cloned into PGEM-T vectors,the two DNA fragments were identified by means of PCR and enzyme evaluation .The results of DNA sequencing showed nucleic acid homogeneity is respectively 99.5% and 99.0% compared with reference gene.Thereby PRRSV simplex RT-PCR and SIV simplex RT-PCR were established .Through optimizing of RT-PCR conditions , the Diagnostic method of multiplex RT-PCR which can simultaneously examine PRRSV and SIV ,was established. 10-4 diluted PRRSV cell culture and SIV chicken embryo allantoic fluid were both detectable by PCR, which is equivalent to 10pg RNA.Twenty six organ samples from different parts of Henan province were respectively detected by multiplex RT-PCR,out of which eight were PRRS positive,four were SI positive, three were both PRRS and SI positive ,control samples were negative. From the experimental outcome,the diagnostic method of multiplex RT-PCR to PRRSV and SIV was rapid(3 5h), specific and sensitive.The method can not only applied to single infection,but to co-infection of PRRSV and SIV as well.
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