Development of a loop-mediated isothermal amplification for rapid detection of orf virus.
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.
Tsai
Su-Ming
SM
Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan.
Chan
Kun-Wei
KW
Hsu
Wei-Li
WL
Chang
Tien-Jye
TJ
Wong
Min-Liang
ML
Wang
Chi-Young
CY
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004474:Ecthyma, Contagious / Q000175:diagnosis*; D005814:Genes, Viral; D006041:Goats; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009923:Orf virus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012867:Skin / Q000821:virology
D017931:DNA Primers