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.
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