Quantitative study of viable Vibrio parahaemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR.
In this study we developed a specific and sensitive quantitative PCR (qPCR) method combined with a propidium monoazide (PMA) sample treatment to quantify tdh-positive viable cells of V. parahaemolyticus in raw seafood (PMA-qPCR). The high selectivity of primers and probes were demonstrated by using purified DNA from 57 strains belonging to 18 species. Using these primers and probes for qPCR and in artificial contamination samples, a good correlation was obtained between Ct values and log CFU/reaction in the range of 12-1.2×10(6)CFU/reaction both from qPCR and PMA-qPCR with R(2) values of 0.9973 and 0.9919, respectively. The optimization of PMA concentration showed that 8 μg/mL was considered optimal to achieve a compromise between minimal impact on intact cells and maximal signal reduction in compromised cells. However, turbidity and cell concentration experiments showed that PMA treatment was not effective in samples where turbidities were ≥10 NTU and OD(600 nm) values were ≥0.8. PMA-qPCR was compared with culture isolation and traditional qPCR in environmental samples (including oyster, scallop, shrimp, and crab). The PMA-qPCR resulted in lower numbers of log CFUg(-1) than qPCR, with values having better agreement with numbers determined by culture isolation. In conclusion, this method is an effective tool for producing reliable quantitative data on viable V. parahaemolyticus in raw seafood.
The Engineering Technology Research Center for Food Biology Processing of Liaoning Province, the Key Laboratory of Food Biology Processing and Quality Control Technology, College of Light Industry, Liaoning University, Shenyang, China. rugangzhu1980@yahoo.com.cn
Ru-Gang
RG
Zhu
Tuo-Ping
TP
Li
You-Feng
YF
Jia
Li-Feng
LF
Song
D000818:Animals; D001386:Azides / Q000737:chemistry*; D003386:Brachyura / Q000382:microbiology; D004396:Coloring Agents / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005516:Food Microbiology; D050296:Microbial Viability; D009391:Nephelometry and Turbidimetry; D010125:Ostreidae / Q000382:microbiology; D049897:Pectinidae / Q000382:microbiology; D033561:Penaeidae / Q000382:microbiology; D011419:Propidium / Q000031:analogs & derivatives* / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000382:microbiology*; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000254:growth & development*
D001386:Azides; D004396:Coloring Agents; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium