Use of sodium lauroyl sarcosinate (sarkosyl) in viable real-time PCR for enumeration of Escherichia coli.
The cell membranes of inactivated Escherichia coli are not always permeable to propidium monoazide (PMA). This limits the use of PMA real-time PCR (PMA-qPCR) for quantification of DNA from only viable cells for enumeration of E. coli. The aim of this study was to develop PMA-qPCR procedures for E. coli with improved selectivity for viable cells. E. coli inactivated by incubation at 52°C were treated with 12 detergents before PMA treatment, and DNA was quantified by real-time PCR. Treatment with each of the 12 detergents and PMA increased the cycle threshold (Ct) values for heat inactivated E. coli suspensions. The greatest increase, of 10.68 Ct was obtained with sarkosyl. Treatment with sodium deoxycholate (NaDC) increased the Ct value by 8.99 Ct. Treatment with sarkosyl or NaDC of 16 heat treated 5-strain cocktails of verotoxigenic E. coli (VTEC) increased the mean Ct values by 8.15 or 6.82 Ct, respectively. Those mean values were significantly (p<0.05) different. When used to enumerate viable E. coli in suspensions treated with lactic acid or in mixtures of viable E. coli and E. coli inactivated by peroxyacetic acid, the slopes relating the Ct values from sarkosyl treated samples to the numbers of viable E. coli were 2.24 and 2.47, respectively, with regression coefficient values ≥0.85. The findings show that sarkosyl was more effective than NaDC for dissipation of PMA-barrier properties of membranes of inactivated E. coli cells. Viable E. coli in mixtures of viable E. coli and E. coli inactivated by heat, lactic acid or peroxyacetic acid could be reliably enumerated by sarkosyl PMA-qPCR.
Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000C & E Trail, Lacombe, Alberta, T4L 1 W1, Canada.
Hui
H
Wang
Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000C & E Trail, Lacombe, Alberta, T4L 1 W1, Canada.
Colin O
CO
Gill
Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000C & E Trail, Lacombe, Alberta, T4L 1 W1, Canada. Electronic address: xianqin.yang@agr.gc.ca.
Xianqin
X
Yang
D001386:Azides / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D003840:Deoxycholic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics*; D019344:Lactic Acid / Q000494:pharmacology; D010463:Peracetic Acid / Q000494:pharmacology; D011419:Propidium / Q000031:analogs & derivatives / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000494:pharmacology
D001386:Azides; D004269:DNA, Bacterial; C533957:propidium monoazide; D003840:Deoxycholic Acid; D019344:Lactic Acid; D011419:Propidium; C025231:sarkosyl; D010463:Peracetic Acid; D012521:Sarcosine
Detergent; Escherichia coli; Propidium monoazide; Sarkosyl; Sodium deoxycholate; Verotoxigenic E. coli