Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses

• Published in: Journal of virological methods Vol. 183; no. 1; pp. 69 - 74
• Main Authors: Xu, Xin-Gang, Chen, Guang-Da, Huang, Yong, Ding, Li, Li, Zhao-Cai, Chang, Ching-Dong, Wang, Chi-Young, Tong, De-Wen, Liu, Hung-Jen
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01.07.2012; Elsevier
• Subjects: Animals; Biological and medical sciences; Classical swine fever virus; Coinfection - diagnosis; Coinfection - veterinary; Coinfection - virology; complementary DNA; CSFV; DNA Primers - genetics; DNA Virus Infections - diagnosis; DNA Virus Infections - veterinary; DNA Virus Infections - virology; DNA viruses; DNA Viruses - genetics; DNA Viruses - isolation & purification; Encephalitis; fetus; Fetuses; Fundamental and applied biological sciences. Psychology; genomics; Hog cholera; Japanese encephalitis virus; JEV; Microbiology; Mixed infection; Multiplex polymerase chain reaction; Multiplex Polymerase Chain Reaction - methods; nucleic acids; PCV2; piglets; Polymerase chain reaction; Porcine circovirus; Porcine circovirus-2; Porcine parvovirus; Porcine pseudorabies virus; Porcine reproductive and respiratory syndrome virus; Porcine respiratory and reproductive syndrome virus; PPV; Primers; PRRSV; PRV; reverse transcriptase polymerase chain reaction; Reverse transcription; RNA; RNA Virus Infections - diagnosis; RNA Virus Infections - veterinary; RNA Virus Infections - virology; RNA viruses; RNA Viruses - genetics; RNA Viruses - isolation & purification; Sensitivity and Specificity; Suid herpesvirus 1; Swine; Swine Diseases - diagnosis; Swine Diseases - virology; Techniques used in virology; Ungulate protoparvovirus 1; Veterinary Medicine - methods; Virology; Virology - methods; viruses; Multiplex polymerase chain reaction; PRV; PPV; JEV; PCV2; PRRSV; CSFV; Method; Vertebrata; Mammalia; Swine; Artiodactyla; JE; Detection; Veterinary; Ungulata
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2012.03.034

► A multiplex polymerase chain reaction (mPCR) assay was developed. ► The mPCR detects simultaneously six DNA or RNA viruses in single reaction. ► The mPCR detects 450pg of six DNA or RNA viruses in single reaction. Uniplex and multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and evaluated subsequently for its effectiveness in detecting simultaneously single and mixed infections in swine. Specific primers for three DNA viruses and three RNA viruses, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive detecting at least 450pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty clinical samples and aborted fetuses collected from 4- to 12-week-old piglets were detected among 39 samples tested by both uniplex and multiplex PCR, showing highly identification. Because of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in swine.