Development and application of loop-mediated isothermal amplification methods targeting the seM gene for detection of Streptococcus equi subsp. equi.
Loop-mediated isothermal amplification (LAMP) constitutes a potentially valuable diagnostic tool for rapid diagnosis of contagious diseases. In this study, we developed a novel LAMP method (seM-LAMP) to detect the seM gene of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles in equids. The seM-LAMP successfully amplified the target sequence of the seM gene at 63°C within 60 min. The sensitivity of the seM-LAMP was slightly lower than the 2nd reaction of the seM semi-nested PCR. To evaluate the species specificity of the seM-LAMP, we tested 100 S. equi and 189 non-S. equi strains. Significant amplification of the DNA originating from S. equi was observed within 60 min incubation, but no amplification of non-S. equi DNA occurred. The results were identical to those of seM semi-nested PCR. To investigate the clinical usefulness of the methods, the seM-LAMP and the seM semi-nested PCR were used to screen 590 nasal swabs obtained during an outbreak of strangles. Both methods showed that 79 and 511 swabs were S. equi positive and negative, respectively, and the results were identical to those of the culture examination. These results indicate that the seM-LAMP is potentially useful for the reliable routine diagnosis of Streptococcus equi subsp. equi infections.
Epizootic Research Center, Equine Research Institute, Japan Racing Association, Shimotsuke-shi, Tochigi 329–0412, Japan. hobo@epizoo.equinst.go.jp
Seiji
S
Hobo
Hidekazu
H
Niwa
Kazuomi
K
Oku
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D006734:Horse Diseases / Q000175:diagnosis / Q000382:microbiology; D006736:Horses; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary*; D013045:Species Specificity; D013290:Streptococcal Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary; D018502:Streptococcus equi / Q000302:isolation & purification* / Q000378:metabolism*; D013997:Time Factors; D014764:Viral Proteins / Q000378:metabolism*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C107891:SeM protein, Streptococcus equi; D014764:Viral Proteins