[Faster detection of Vibrio parahaemolyticus in foods by FQ-PCR technique].
OBJECTIVE
To establish a rapid and accurate qualitative and quantitative method to detect Vibrio parahaemolyticus in food.


METHODS
Primers and Taqman probe were designed according to the sequence of gyrase gene of Vibrio parahaemolyticus. The PCR fragment was cloned into PTM vector and was used as positive template for establishing the criterion curve. The simulated samples, made from negative food samples with Vibrio parahaemolyticus positive strain, were used to evaluate the sensitivity of the PCR reaction. 8 Vibrio parahaemolyticus standard strains and other 24 negative strains (8 strains were Vibrionaceae other than Vibrio parahaemolyticus, the rest 16 strains were none-Vibrionaceae) were treated in the same way to evaluate the specificity. All samples were detected with PE7000 sequence detection system and DA620 fluorescene detection system.


RESULTS
FQ-PCR method had good specificity and high sensitivity. All 8 strains of Vibrio parahaemolyticus tested showed positive results while all other 24 strains were negative (8 strains were Vibrionaceae, 16 strain were from different). The correlation was 0.9871 between the concentration of positive template and the quantitative curve circular threshold. The threshold for detecting Vibrio parahaemolyticus in pure culture is 10 cfu/ml, and the threshold is as low as 2 cfu/ml with 16 simulative samples after these samples were incubated for a period of time. By direct quantitative detection for uncultured 16 food samples, the threshold is 100 cfu/g.


CONCLUSIONS
FQ-PCR method has high sensitivity and specificity, and it is a handy and rapid detection method. Comparing with regular PCR method, it is not easily contaminated in operation, and can achieve high sensitivity and specificity with domestic instruments. FQ-PCR method has potential and applied value for detection of pathogenic bacteria in food.
Jiao
Hong
H
Food Inspection Center of Guangzhou Quarantine Bureau of People's Republic of China, Guangzhou 510623, China.
Weng
Wen-Chuan
WC
Wang
Fang-Jin
FJ
Cheng
Gang
G
Wang
Weiyi
W
Xie
Junxian
J
D000818:Animals; D005399:Fishes / Q000382:microbiology; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014736:Vibrio parahaemolyticus / Q000302:isolation & purification*