title: Development of a loop-mediated isothermal amplification assay for the detection of porcine hokovirus.
abstract: Hokoviruses have recently been detected as pathogens belonging to the family Parvoviridae, which comprises porcine hokovirus (PHoV) and bovine hokovirus (BHoV). In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific and sensitive detection of PHoV. A set of four primers specific for six regions within the PHoV VP1/2 genes was designed using online software. The reaction temperature and time were optimized at 65°C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change caused by a fluorescent dye. The method was highly specific for PHoV, and no cross-reaction was observed with porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine bocavirus (PBoV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The detection limit was approximately 10 copies per reaction, which was 10 times more sensitive than conventional PCR. Furthermore, the efficiency of detection of PHoV in clinical samples was comparable to that of PCR and sequencing. These results show that the LAMP assay is a simple, rapid, sensitive and specific method for detecting PHoV. It does not require specialized equipment and can be used to detect PHoV both in the laboratory and in the field.
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences; Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture; National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, Jiangsu Province, China. Electronic address: libinana@126.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: Bing
authors.initials: B
authors.lastname: Sun
authors.forename: Lu-ping
authors.initials: LP
authors.lastname: Du
authors.forename: Ai-hua
authors.initials: AH
authors.lastname: Mao
authors.forename: Li-bin
authors.initials: LB
authors.lastname: Wen
authors.forename: Yan-xiu
authors.initials: YX
authors.lastname: Ni
authors.forename: Xue-han
authors.initials: XH
authors.lastname: Zhang
authors.forename: Kong-wang
authors.initials: KW
authors.lastname: He
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010322:Parvoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D019219:Parvovirinae / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D013696:Temperature; D013997:Time Factors; D014730:Veterinary Medicine / Q000379:methods*; D015678:Viral Structural Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D015678:Viral Structural Proteins
keywords: Detection; Loop-mediated isothermal amplification; PCR; Porcine hokovirus