title: A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.
abstract: Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections.
authors: Beijing Entry-Exit Inspection & Quarantine Bureau, Beijing, China; Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States.
authors: Xiju
authors: X
authors: Shi
authors: Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States.
authors: Xuming
authors: X
authors: Liu
authors: China Institute of Veterinary Drug Control, Beijing, China.
authors: Qin
authors: Q
authors: Wang
authors: Foreign Animal Diseases Diagnostic Laboratory, NVSL, APHIS, USDA, Greenport, NY, United States.
authors: Amaresh
authors: A
authors: Das
authors: Beijing Entry-Exit Inspection & Quarantine Bureau, Beijing, China.
authors: Guiping
authors: G
authors: Ma
authors: China Institute of Veterinary Drug Control, Beijing, China.
authors: Lu
authors: L
authors: Xu
authors: Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States.
authors: Qing
authors: Q
authors: Sun
authors: Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States.
authors: Lalitha
authors: L
authors: Peddireddi
authors: Foreign Animal Diseases Diagnostic Laboratory, NVSL, APHIS, USDA, Greenport, NY, United States.
authors: Wei
authors: W
authors: Jia
authors: Beijing Entry-Exit Inspection & Quarantine Bureau, Beijing, China.
authors: Yanhua
authors: Y
authors: Liu
authors: Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States.
authors: Gary
authors: G
authors: Anderson
authors: Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States. Electronic address: jbai@vet.ksu.edu.
authors: Jianfa
authors: J
authors: Bai
authors: Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States. Electronic address: jshi@ksu.edu.
authors: Jishu
authors: J
authors: Shi
mesh_terms: D000818:Animals; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D014780:Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
keywords: Diagnostic; Panel assay; RT-PCR; Real-time PCR; Swine viruses