title: Bead-based suspension array for simultaneous differential detection of five major swine viruses.
abstract: A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2-22 copies/μL (corresponding to 0.5-6.8 fg/μL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen.
authors.affiliation: Animal Inspection and Quarantine Laboratory, Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, No.66 Huacheng Dadao Ave., Zhujiangxincheng, Guangzhou, 510623, China, gd_chenr@sina.com.
authors.forename: Ru
authors.initials: R
authors.lastname: Chen
authors.forename: Xiao-Lu
authors.initials: XL
authors.lastname: Yu
authors.forename: Xiao-Bo
authors.initials: XB
authors.lastname: Gao
authors.forename: Cun-Yi
authors.initials: CY
authors.lastname: Xue
authors.forename: Chang-Xu
authors.initials: CX
authors.lastname: Song
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Yong-Chang
authors.initials: YC
authors.lastname: Cao
mesh_terms: D000818:Animals; D017925:Circovirus / Q000145:classification / Q000302:isolation & purification*; D006691:Classical Swine Fever / Q000175:diagnosis / Q000821:virology; D006692:Classical Swine Fever Virus / Q000302:isolation & purification*; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction; D028323:Parvovirus, Porcine / Q000302:isolation & purification*; D019316:Porcine respiratory and reproductive syndrome virus / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013552:Swine / Q000821:virology; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral