Isolation of partial toxR gene of Vibrio harveyi and design of toxR-targeted PCR primers for species detection

Abstract

Aims: To differentiate Vibrio harveyi from closely related Vibrio species by toxR sequence analysis and design primers for the specific detection of the shellfish pathogen. Methods and Results: The partial toxR homologue from the shellfish pathogen V. harveyi was isolated by PCR using degenerate primers. The 578-bp toxR fragment from V. harveyi, that exhibited highest homology with partial toxR of V. parahaemolyticus (68%), is predicted to encode for a polypeptide with 192 amino acid residues. Alignment of the V. harveyi toxR nucleotide and deduced amino acid sequence with those from other Vibrio species revealed the presence of the fairly characteristic conserved transcription activation and transmembrane domain as well as the divergent membrane tether region that may be targeted for the development of species-specific oligonucleotide primers. Consequently, PCR primers that could amplify a 390-bp gene fragment in V. harveyi were designed by targeting portions of the V. harveyi toxR that display variability with toxR sequences from other Vibrio species. The 390-bp-amplicon was detected in all V. harveyi strains examined except in the nontarget bacteria and unexpectedly, in two shrimp-derived strains (VIB 391 and STD 3-101) from Thailand and Ecuador. Results show that strains exhibiting the 390-bp amplicon mostly belong to the same cluster based on previous amplified fragment length polymorphism data while strains which were previously unclustered or unclassified did not display the 390-bp PCR product. Conclusions: The toxR sequence variation could differentiate V. harveyi from closely related Vibrio species. A PCR protocol amplifying a 390-bp fragment of the V. harveyi toxR was established and could be useful in the specific and rapid detection of the species. Significance and Impact of the Study: The molecular approaches reported in this study could facilitate the early diagnosis and surveillance of luminous vibriosis in hatchery-reared fish and shellfish species through rapid identification and specific detection of causal agent.

Amplification and sequence analysis of the full length toxR gene in Vibrio harveyi

“…In addition to identification, and as described in an earlier study (Conejero and Hedreyda, 2003), the hypervariable region of toxR could be targeted in primer design for species detection. The availability of the complete toxR sequence of V. harveyi is necessary in order to evaluate the usefulness of toxR as a gene marker for species detection and classification.…”

“…Gene sequences for toxR have been amplified from V. alginolyticus, V. mimicus, V. fluvialis and V. hollisae (Gutacker et al, 2003;Osorio and Klose, 2000;Vuddhakul et al, 2000). Conejero and Hedreyda (2003) were able to isolate and sequence a 576-bp toxR gene homologue from V. harveyi (NBRC 15634). This paper reports the design and selection of primers to amplify regions of the V. harveyi toxR gene flanking the 576-bp internal fragment.…”

Differential detection of vibrios pathogenic to shrimp by multiplex PCR

“…The use of the newly designed primers (hemolysintargeted primers for detecting Philippine Vibrio isolates and toxR-targeted PCR primers for specific detection of type strain V. campbellii NBRC 15631) in this study, in combination with the toxR-targeted primer specific for the detection of V. harveyi (Conejero and Hedreyda, 2003), will be useful in differential detection of type strain-like V. harveyi, type strain-like V. campbellii, and strains exhibiting characteristics similar to the 10 variant Philippine Vibrio isolates used in this study. PCR profiles obtained in this study reveal (Fig.…”

“…4) were generated depending on what DNA template was added (DNA template from type strain V. harveyi, type strain V. campbellii, or variant Philippine Vibrio isolates). In multiplex PCR, the PNhemo, VcatoxR, and VhtoxR (Conejero and Hedreyda, 2003) primers specifically amplified a 320-bp Philippine Vibrio isolate hemolysin gene fragment (Fig. 4, lanes 3-13), a 245-bp toxR fragment from type strain V. campbellii (Fig.…”

Sequence analysis of partial toxR gene from Philippine Vibrio isolates and design of toxR-targeted primers for detection

“…PCR methods targeting toxR sequence variations have been utilized for the detection and isolation of Vibrio species (Kim et al, 1999;Vuddhakul et al, 2000). We have developed in our previous studies toxR-targeted PCR primers specific for the detection of Vibrio harveyi from different geographic regions (Conejero and Hedreyda, 2003). We have also used PCR primers based on toxR sequences of type strain Vibrio campbellii for its specific detection (San Luis and Hedreyda, 2005).…”