Establishment of a porcine parvovirus (PPV) DNA standard and evaluation of a new lightcycler nested-PCR assay for detection of PPV.
Porcine parvovirus (PPV) is a major causative agent in a syndrome of reproductive failure in swine. In validation (viral clearance) studies, PPV is a model of non-enveloped viruses and is widely used instead of human parvovirus B19, which causes a variety of illnesses including erythema infectiosum (fifth disease) in children and hydrops fetalis in pregnant women. To improve the sensitivity of current PCR-based assays for detection of PPV and to standardize the quantification of PPV, we have developed a lightcycler (LC) nested-PCR (nPCR) assay and constructed a PPV DNA standard evaluated in the LC nPCR assay. The PPV DNA standard, a plasmid termed pPPV, encodes a 3.3 kb PPV NADL-2 genome fragment. One genome copy equivalent (gce) of PPV equals 6.7 attograms of pPPV. The LC nPCR assay is a simple and specific method developed for detection of PPV strains but not any other viruses including members of Parvoviridae. The first 25-cycle PCR with outer primers chose by comparative analysis of 12 primers in 21 different combinations and a second 45-cycle PCR with inner primers amplify 286 and 251 bp fragments of PPV genome, respectively, for 40 min with a sensitivity of approximately 100 gce per assay (ml). By using the LC nPCR assay for analysis of PPV samples with known infectivity, we found that one 50% tissue culture infectious dose (TCID(50)) equals 1.93 +/- 0.24 log(10) gce.
Prikhod'ko
Grigori G
GG
Plasma Derivatives Department, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA. prikhodkog@usa.redcross.org
Reyes
Herbert
H
Vasilyeva
Irina
I
Busby
Thomas F
TF
D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000592:standards; D005069:Evaluation Studies as Topic; D016679:Genome, Viral; D028323:Parvovirus, Porcine / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D016415:Sequence Alignment; D013045:Species Specificity; D013552:Swine
D017931:DNA Primers; D004279:DNA, Viral