Comprehensive and rapid real-time PCR analysis of 21 foodborne outbreaks

Abstract

A set of four duplex SYBR Green I PCR (SG-PCR) assay combined with DNA extraction using QIAamp DNA Stool Mini kit was evaluated for the detection of foodborne bacteria from 21 foodborne outbreaks. The causative pathogens were detected in almost all cases in 2 hours or less. The first run was for the detection of 8 main foodborne pathogens in 5 stool specimens within 2 hours and the second run was for the detection of other unusual suspect pathogens within a further 45 minutes. After 2 to 4 days, the causative agents were isolated and identified. The results proved that for comprehensive and rapid molecular diagnosis in foodborne outbreaks, Duplex SG-PCR assay is not only very useful, but is also economically viable for one-step differentiation of causative pathogens in fecal specimens obtained from symptomatic patients. This then allows for effective diagnosis and management of foodborne outbreaks.

Figure 1

Melting curve analysis of duplex SYBR Green I PCR products in the first run using four primer sets: FemB plus eaeA, AB plus EAST1, ces plus tdh, and GAP plus Styinva.

Figure 2

Melting curve analysis of duplex SYBR Green I PCR products in the second run using four primer sets: ST plus PSG, aggR plus virA, LT plus AHH1, and PAG plus SG; the third run using two primer sets: CCcesE plus yadA and trh plus hlyA; simple PCR with primers JMS 1 and JMS2.

Figure 3

The relationship between CFU and DNA copy of foodborne pathogens in 71 foodborne pathogens-positive feces in 14 foodborne outbreak cases examined by viable cell counting.