Detection of Vibrio parahaemolyticus in tropical shellfish by SYBR green real-time PCR and evaluation of three enrichment media.
A rapid, sensitive and highly reproducible SYBR green based real-time PCR assay was developed for detection of tdh positive pathogenic Vibrio parahaemolyticus. Minimum detection limit was 0.1 pg of pure V. parahaemolyticus genomic DNA with typical R(2) values >0.99 and coefficient of variation (CV) values ranging from 1.2 to 4.2 on three different days. The method was also used to evaluate the effect of three different enrichment media alkaline peptone water (APW), sodium taurocholate (ST) broth and salt polymyxin broth (SPB) on detection of V. parahaemolyticus. Crude lysates were directly used for real-time PCR. Without enrichment, the detection limit of pure cultures was 10(1) CFU/ml for ST broth and 10(2) CFU/ml in APW and SPB but for shrimp homogenates spiked with pure culture, the minimum detection limit was 10(2) CFU/ml for all three broths with a linear detection range of 10(2)-10(6). Without enrichment, detection in ST broth was more efficient than APW and SPB. After 6 h enrichment, limit of detection was found to be 1 CFU in all three media. However, for iced shrimp, the limit of detection was 10(2) after 6 h enrichment. No significant difference was seen between different enrichment media with respect to tdh gene detection of V. parahaemolyticus. The methodology developed here can be useful for rapid detection of tdh positive V. parahaemolyticus by laboratories involved in monitoring programmes for pathogenic V. parahaemolyticus.
Tyagi
Anuj
A
Department of Fishery Microbiology, Karnataka Veterinary, Animal and Fisheries Sciences University, College of Fisheries, Mangalore-575 002, India.
Saravanan
V
V
Karunasagar
Iddya
I
Karunasagar
Indrani
I
D000818:Animals; D001427:Bacterial Toxins; D015169:Colony Count, Microbial; D003470:Culture Media / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes; D005506:Food Contamination / Q000032:analysis*; D006460:Hemolysin Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000382:microbiology*; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity
D001427:Bacterial Toxins; D003470:Culture Media; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; C068489:thermostable direct hemolysin