Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp

Abstract

Because biochemical testing and 16S rRNA sequence analysis have proven inadequate for the differentiation of Vibrio parahaemolyticus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic probe. The gyrB genes of V. parahaemolyticus and closely related Vibrio alginolyticus were cloned and sequenced. Oligonucleotide PCR primers were designed for the amplification of a 285-bp fragment from within gyrB specific for V. parahaemolyticus. These primers recognized 117 of 117 reference and wild-type V. parahaemolyticus strains, whereas amplification did not occur when 90 strains of 37 other Vibrio species or 60 strains representing 34 different nonvibrio species were tested. In 100-microliter PCR mixtures, the lower detection limits were 5 CFU for live cells and 4 pg for purified DNA. The possible application of gyrB primers for the routine identification of V. parahaemolyticus in food was examined. We developed and tested a procedure for the specific detection of the target organism in shrimp consisting of an 18-h preenrichment followed by PCR amplification of the 285-bp V. parahaemolyticus-specific fragment. This method enabled us to detect an initial inoculum of 1.5 CFU of V. parahaemolyticus cells per g of shrimp homogenate. By this approach, we were able to detect V. parahaemolyticus in all of 27 shrimp samples artificially inoculated with this bacterium. We present here a rapid, reliable, and sensitive protocol for the detection of V. parahaemolyticus in shrimp.

FIG. 1

Nucleotide sequence of gyrB of V. parahaemolyticus ATCC 17802 and V. alginolyticus ATCC 17749. Nucleotides identical to those of V. parahaemolyticus are indicated with dots.

FIG. 2

Amino acid sequence alignment of the gyrB products from V. parahaemolyticus ATCC 17802 and V. alginolyticus ATCC 17749. Amino acids identical to those of V. parahaemolyticus are indicated with dots.

FIG. 3

Sensitivity of VP-1 and VP-2r PCR primers for the amplification of the V. parahaemolyticus-specific amplicon at various DNA concentrations. DNAs were extracted from overnight cultures in APW by phenol-chloroform extraction and ethanol precipitation. DNA concentrations were measured with a spectrophotometer, and DNA was serially diluted in TE buffer to obtain the appropriate concentrations. Lanes contained V. parahaemolyticus DNA (unless otherwise noted): M, 100-bp DNA ladder; 1, 4.3 ng; 2, 430 pg; 3, 43 pg; 4, 4 pg; 5, 430 fg; 6, V. alginolyticus DNA (10 ng).

FIG. 4

Sensitivity of VP-1 and VP-2r PCR primers for the amplification of the V. parahaemolyticus-specific amplicon at various bacterial concentrations. Bacterial cells were grown in APW for 18 h at 37°C and serially diluted in PBS containing 2% NaCl. Appropriate dilutions were spread plated on marine agar, and bacterial counts were enumerated after 18 h of incubation at 37°C. Lanes contained V. parahaemolyticus (unless otherwise noted): M, 100-bp DNA ladder; 1, 5.2 × 10³ CFU per reaction tube; 2, 5.2 × 10² CFU per reaction tube; 3, 5.2 × 10¹ CFU per reaction tube; 4, 5.2 CFU per reaction tube; 5, PCR mixture control without any added bacterial cells; 6, V. alginolyticus bacterial cells (7.9 × 10³ CFU per reaction tube).

FIG. 5

Detection of V. parahaemolyticus in artificially contaminated shrimp by the VP-1 and VP-2r PCR primers. V. parahaemolyticus cells grown overnight in APW were serially diluted in shrimp homogenate (see details in Materials and Methods) to obtain the appropriate dilutions. Total microflora of shrimp were counted in marine agar (48 h), and the V. parahaemolyticus population was counted in TCBS agar (18 h) after incubation at 37°C. Lanes: M, 100-bp DNA ladder; 1, shrimp homogenate not spiked with V. parahaemolyticus cells; 2, initial inoculum of 1.5 CFU of V. parahaemolyticus per g added to shrimp homogenate and incubated for 18 h at 37°C; 3, initial inoculum of 1.5 × 10⁵ CFU of V. parahaemolyticus per g added to shrimp homogenate sampled at zero hour; 4, 1.5 CFU of V. parahaemolyticus cells prepared in PCR mixture.