title: Novel multiplex PCR assay using locked nucleic acid (LNA)-based universal primers for the simultaneous detection of five swine viruses.
abstract: A novel multiplex PCR assay using non-homologous oligonucleotides with locked nucleic acid (LNA) modifications as universal primers was developed and validated for the simultaneous detection of five swine viruses. The assay utilizes five virus-specific primer pairs modified at the 5' end through the addition of the universal primer sequence. In the reaction, small amounts of target templates with the 5' tail were generated and subsequently amplified through the extension of a LNA universal primer set. To validate the specificity of this assay, 27 viral target strains and 12 non-target pathogens were tested. The lower limit of detection of viral nucleic acids was 1.1-1.9 pg per reaction or 11-32 pg in a five-plex viral nucleic acid mixture. The LNA mPCR assay displayed higher analytical sensitivity and efficiency for the detection of plasmid standards compared with the conventional assay, which uses standard primers without the 5' tail. A total of 207 field samples were tested using both assays. The LNA mPCR assay provided numerically higher detection rates for all pathogens in independent samples. Moreover, the LNA mPCR assay had significantly higher detection rates in independent samples compared with the conventional assay.
authors.affiliation: Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou 510623, China. Electronic address: gd_chenr@sina.com.
authors.forename: Ru
authors.initials: R
authors.lastname: Chen
authors.affiliation: Department of Genetics, National Research Institute for Family Planning, Beijing 100081, China.
authors.forename: Xiao-Bo
authors.initials: XB
authors.lastname: Gao
authors.affiliation: School of Life Sciences, Sun Yat-Sen University, Guangzhou 510675, China.
authors.forename: Xiao-Lu
authors.initials: XL
authors.lastname: Yu
authors.affiliation: Veterinary Medicine Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China.
authors.forename: Chang-Xu
authors.initials: CX
authors.lastname: Song
authors.affiliation: Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou 510623, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Qiu
mesh_terms: D000818:Animals; D017925:Circovirus / Q000235:genetics / Q000302:isolation & purification; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000302:isolation & purification; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009841:Oligonucleotides; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D014777:Virus Diseases / Q000662:veterinary*; D014780:Viruses / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D009841:Oligonucleotides; C477371:locked nucleic acid
keywords: Locked nucleic acid (LNA); Multiplex PCR (mPCR); Swine virus; Universal primer