title: Comparative evaluation of conventional polymerase chain reaction (PCR), with loop-mediated isothermal amplification and SYBR green I-based real-time PCR for the quantitation of porcine circovirus-1 DNA in contaminated samples destined for vaccine production.
abstract: Porcine circovirus type1 (PCV1), described initially as a contaminant of a porcine kidney cell line, is ubiquitous within the swine population The presence of PCV1 in porcine cell lines can lead to contamination during both human and porcine vaccine production. Therefore, a rapid, specific, sensitive and practical method is needed for the detection of PCV1 in bio-products. The aim of this study was to compare three assays in their ability to accurately quantify PCV1 virus in biological samples, namely loop-mediated isothermal amplification (LAMP), SYBR green I-based real-time polymerase chain reaction (PCR) and conventional PCR. All assays yielded successful quantitation of PCV1 DNA and differentiated between PCV1-free and-contaminated cells. In addition, the results were specific for PCV1, since amplification of samples containing closely-related PCV2 or other pathogenic swine viruses yielded negative results. The lowest detection threshold of 10(2) copies was displayed by the SYBR green I-based real-time PCR assay. In addition, this assay was the most effective in detecting PCV1 contamination in a set of commercially available porcine vaccines. Therefore we conclude that SYBR green I-based real-time PCR is specific and sensitive for detecting PCV1 in biological samples and maybe used for quality control of vaccine and biomaterial production.
authors.affiliation: State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences CAAS, No 4899 Juye Street, Chuangchun 130112, China.
authors.forename: Bo-Chao
authors.initials: BC
authors.lastname: Yang
authors.forename: Feng-Xue
authors.initials: FX
authors.lastname: Wang
authors.forename: Shu-Qin
authors.initials: SQ
authors.lastname: Zhang
authors.forename: Ni
authors.initials: N
authors.lastname: Song
authors.forename: Jian-Xi
authors.initials: JX
authors.lastname: Li
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Yang
authors.forename: Yong-Jun
authors.initials: YJ
authors.lastname: Wen
authors.forename: Hua
authors.initials: H
authors.lastname: Wu
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002460:Cell Line; D017925:Circovirus / Q000302:isolation & purification*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013552:Swine; D019562:Viral Load / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I