Species-specific PCR detection of the fish pathogen, Vibrio anguillarum, using the amiB gene, which encodes N-acetylmuramoyl-L-alanine amidase.
Vibrio anguillarum is the causative agent of the fish disease vibriosis and is the most intensely studied species of Vibrio. In the present study, specific primers and a PCR assay were designed to detect V. anguillarum. The primers were designed to amplify a 429-bp internal region of the V. anguillarum amiB gene, which encodes the peptidoglycan hydrolase N-acetylmuramoyl-L-alanine amidase. PCR specificity was demonstrated by successful amplification of DNA from V. anguillarum and by the absence of a PCR product from 25 other Vibrio strains and various enteric bacteria. The PCR produced a 429-bp amplified fragment from as little as 1 pg of V. anguillarum DNA. The limit of detection for this PCR technique was c. 20 bacterial colonies in 25 mg of infected flounder tissue. These results suggest that this PCR system is a sensitive and species-specific detection method, and is possible to use as a diagnostic tool to detect V. anguillarum.
Hong
Gyeong-Eun
GE
Department of Biotechnology and Bioengineering, Pukyong National University, Busan, Korea.
Kim
Dong-Gyun
DG
Bae
Ju-Yoon
JY
Ahn
Sun-Hee
SH
Bai
Sungchul C
SC
Kong
In-Soo
IS
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D005393:Fish Diseases / Q000382:microbiology*; D005432:Flounder / Q000382:microbiology; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D014733:Vibrio / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D014735:Vibrio Infections / Q000382:microbiology / Q000662:veterinary*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D009238:N-Acetylmuramoyl-L-alanine Amidase