Differential detection of vibrios pathogenic to shrimp by multiplex PCR

• Published in: Journal of general and applied microbiology Vol. 52; no. 5; pp. 273 - 280
• Main Authors: Castroverde, Christian Danve M., Luis, Boris B. San, Monsalud, Rosario G., Hedreyda, Cynthia T.
• Format: Journal Article
• Language: English
• Published: Tokyo Applied Microbiology, Molecular and Cellular Biosciences Research Foundation 2006; Microbiology Research Foundation; Japan Science and Technology Agency
• Subjects: Animals; Bacterial Proteins - genetics; Biological and medical sciences; Decapoda; Decapoda (Crustacea) - virology; DNA-Binding Proteins - genetics; Fundamental and applied biological sciences. Psychology; hemolysin; Marine; Microbiology; Polymerase Chain Reaction - methods; toxR; Transcription Factors - genetics; Vibrio; Vibrio - isolation & purification; Vibrio campbellii; Vibrio harveyi; vibriosis; toxR; Vibriosis; Macrura; Crustacea; Infection; Polymerase chain reaction; Analysis method; Vibrio harveyi; Arthropoda; Pathogenic; Vibrio campbellii; Bacteriosis; Bacteria; Vibrionaceae; Decapoda; Invertebrata; Detection; Hemolysin; Shrimp
• ISSN: 0022-1260; 1349-8037
• DOI: 10.2323/jgam.52.273

The research was focused on the multiplex polymerase chain reaction (PCR) differential detection of shrimp pathogens Vibrio harveyi, Vibrio campbellii and isolates from a variant strain of Vibrio (referred to as Philippine Vibrio isolates in this study) exhibiting characteristics distinct from these two species. Sequence alignment of the hemolysin gene from type strains Vibrio harveyi (NBRC 15634) and Vibrio campbellii (NBRC 15631), as well as 10 variant Philippine Vibrio isolates, was performed in order to design a set of hemolysin-targeted primers for the specific detection of the Philippine Vibrio isolates. Primer PNhemo amplified a 320-bp hemolysin gene fragment of the Philippine Vibrio isolates in PCR using 65°C annealing temperature, but did not amplify the target gene fragment in type strains V. harveyi and V. campbellii. Another new primer (VcatoxR) targeting the toxR gene was designed for the specific detection of type strain V. campbellii under stringent 65°C annealing temperature. PCR using VcatoxR primer resulted in the specific amplification of a 245-bp V. campbellii toxR fragment. The simultaneous use of three primer sets in PCR, including PNhemo and VcatoxR (the two new primers designed in this study), and a primer VhtoxR (previously reported for the specific detection of V. harveyi), resulted in differential profiles with 390-bp, 245-bp, and 320-bp amplicons for V. harveyi, V. campbellii, and variant Philippine Vibrio isolates, respectively. Presence of all three types of Vibrio shrimp pathogens in the sample could be detected with a multiplex PCR profile containing all the expected size amplicons.