Simultaneous Quantification by Real-time PCR of Viable Escherichia coli and E. coli O157:H7 in Beef after Heat Treatment

Introduction: Bacteria can be rapidly quantified by determination of DNA from them using real-time PCR (RT-PCR). However, RT-PCR, when used alone, cannot differentiate DNA from live and dead bacteria. We have developed a SYBR green-based RT-PCR procedure in which cells are treated with sodium deoxycholate (SD) and propidium monoazide (PMA) prior to DNA extraction to prevent the amplification of DNA from dead cells with intact and damaged cell membranes. However, the procedure can only quantify one target organism at a time.  

Purpose: To develop a RT-PCR procedure for simultaneous quantification of viable E. coli and E. coli O157:H7 in beef after heat treatment.

Methods: Optimal concentrations of primers and probes targeting the tuf gene and rfbE gene in E. coli and E. coli O157:H7, respectively, and a 79-bp synthetic nucleotide fragment used as an internal amplification control (IAC), were determined by comparing the amplification efficiencies resulting from different concentrations. The ubiquity of tuf gene in E. coli was tested against a total of 118 strains of E. coli from beef (97 non O157 and 21 O157). The optimized triplex tuf/rfbE/IAC assay in conjunction with SD and PMA was used to determine the surviving E. coli and E. coli O157:H7 in meat juice heated at 52°C.

Results: All 118 strains of E. coli were positive for the tuf gene. The triplex assay was able to quantify DNA templates of E. coli and E. coli O157:H7 ranging from 10⁶ to 5x10¹ with amplification efficiencies between 96.8% and 103.0%. After heating at 52°C, cycle threshold (Ct) values, determined by RT-PCR, increased with decreasing viable cell numbers, determined by plating, at 1.78 and 1.86 Ct/log CFU for E. coli and E. coli O157:H7, respectively.

Significance: The developed triplex RT-PCR procedure could be used for simultaneous quantification of viable E. coli and E. coli O157:H7 in beef that has gone through heat treatment.