title: Rapid detection of orf virus by loop-mediated isothermal amplification based on the DNA polymerase gene.
abstract: At present, there are no effective antiviral treatments available for contagious ecthyma, and rapid diagnosis is therefore critical for effective control of the disease. Recently, the invention of a novel LAMP technique that can rapidly amplify nucleic acids with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic acid-based diagnostic tests and has made on-site diagnosis possible. To establish a flexible loop-mediated isothermal amplification (LAMP) assay for the rapid detection of orf virus, two pairs of primers, including outer primers F3/B3 and inner primers FIP/BIP, were designed to amplify the DNA polymerase gene. Optimal time and temperature conditions for LAMP were found to be 45 min and 62 °C, respectively. The LAMP assay was shown to be specific, with no cross-reactivity with sheeppox virus, goatpox virus, avian molluscum roup virus or vesicular stomatitis virus. Additionally, the sensitivity of the LAMP method was similar to that of real-time PCR and demonstrated greater sensitivity than a conventional polymerase chain reaction (PCR) assay. To assess the utility of LAMP in the detection of orf virus in clinical samples, a total of 35 samples collected from orf virus-infected sheep and goats were tested using the optimized LAMP assay, real-time PCR, and conventional PCR. Of the samples, 26 were found to be positive by LAMP, and 25 (74.3 %) were positive by real-time PCR, whereas only 18 (51.4 %) were positive by conventional PCR. Our results have shown that the LAMP assay developed in this study can be used for the rapid detection of orf virus.
authors.affiliation: College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi'an road, Changchun, 130062, China.
authors.forename: Jida
authors.initials: J
authors.lastname: Li
authors.forename: Deguang
authors.initials: D
authors.lastname: Song
authors.forename: Wenqi
authors.initials: W
authors.lastname: He
authors.forename: Yingfu
authors.initials: Y
authors.lastname: Bao
authors.forename: Rongguang
authors.initials: R
authors.lastname: Lu
authors.forename: Gaoli
authors.initials: G
authors.lastname: Su
authors.forename: Gaili
authors.initials: G
authors.lastname: Wang
authors.forename: Huijun
authors.initials: H
authors.lastname: Lu
authors.forename: Kui
authors.initials: K
authors.lastname: Zhao
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
mesh_terms: D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D004474:Ecthyma, Contagious / Q000175:diagnosis / Q000821:virology*; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D021141:Nucleic Acid Amplification Techniques; D009923:Orf virus / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D012756:Sheep
chemical_list: D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase