Rapid quantification of Vibrio vulnificus in clams (Protochaca staminea) using real-time PCR.
We used a rapid DNA extraction and purification method to obtain the DNA from Vibrio vulnificus seeded into clam tissue homogenates for real-time PCR quantification of the organism. Without enrichment, the limit of detection was 1 x 10(2) cfu/g of tissue with a linear detection range of 1 x 10(2) to 1 x 10(8) cfu/g. With a 5 h non-selective enrichment, the limit of detection was 1 cfu/g of tissue with a linear detection range of 1 to 1 x 10(6) cfu/g of tissue. We found a 10-fold higher detection limit with seeded clam tissue homogenates compared to pure culture in TSB(+). The detection limits with pure broth culture and seeded tissue homogenates were identical, 1 cfu/ml and 1 cfu/ml, respectively, following 5 h non-selective enrichment. However, the Ct value with tissue homogenates was about 3 threshold cycles higher than with pure culture.
Wang
Shishan
S
Department of Food Science, Massachusetts Agricultural Experiment Station, University of Massachusetts, Amherst, 01003, USA.
Levin
Robert E
RE
D000818:Animals; D049872:Bivalvia / Q000382:microbiology*; D015169:Colony Count, Microbial / Q000379:methods; D003257:Consumer Product Safety; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012758:Shellfish / Q000382:microbiology*; D013997:Time Factors; D041261:Vibrio vulnificus / Q000302:isolation & purification*
D003470:Culture Media; D004269:DNA, Bacterial