Development of a loop-mediated isothermal amplification for rapid detection of orf virus

• Published in: Journal of virological methods Vol. 157; no. 2; pp. 200 - 204
• Main Authors: Tsai, Su-Ming, Chan, Kun-Wei, Hsu, Wei-Li, Chang, Tien-Jye, Wong, Min-Liang, Wang, Chi-Young
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01.05.2009; Elsevier
• Subjects: Animals; Base Sequence; Biological and medical sciences; DNA Primers - genetics; Ecthyma, Contagious - diagnosis; Fundamental and applied biological sciences. Psychology; Genes, Viral; Goats; Loop-mediated isothermal amplification (LAMP); Microbiology; Molecular Sequence Data; Nested PCR; Nucleic Acid Amplification Techniques - methods; Orf virus; Orf virus - genetics; Orf virus - isolation & purification; PCR; Sensitivity and Specificity; Skin - virology; Techniques used in virology; Virology; orf virus; Nested PCR; Loop-mediated isothermal amplification (LAMP); PCR; Chordopoxvirinae; Nested polymerase chain reaction; Microbiology; Loop-mediated isothermal amplification; Method; Orf virus; Virology; Virus; Amplification; Polymerase chain reaction; Poxviridae; LAMP; Detection; Parapoxvirus
• ISSN: 0166-0934; 1879-0984; 1879-0984
• DOI: 10.1016/j.jviromet.2009.01.003

A loop-mediated isothermal amplification (LAMP) assay using six primers targeting a highly conserved region of the B2L gene has been developed to diagnose orf virus. The assay produces a ladder-like pattern of products on an agarose gel that can be specifically digested with BsrGI enzyme. The sensitivity of the LAMP assay, which was determined to be a single copy of the standard plasmid, was 100 fold and 10 fold higher than PCR and nested PCR, respectively; furthermore, no cross-reactivity was founded with the other tested viruses. By staining the products directly in the tube with PicoGreen or ethidium bromide, the products can be visualized with a similar sensitivity as by gel electrophoresis. Clinical samples were tested using PCR, nested PCR and LAMP assay and the positive rates were 60%, 70% and 70%, respectively. The LAMP assay allows easy, rapid, accurate and sensitive detection of infection with orf virus and is especially applicable in a resource-limited situation.