Homogenous, real-time duplex loop-mediated isothermal amplification using a single fluorophore-labeled primer and an intercalator dye: Its application to the simultaneous detection of Shiga toxin genes 1 and 2 in Shiga toxigenic Escherichia coli isolates

Jiang

Yang

et al. 2012

Appl Environ Microbiol

ABSTRACTEscherichia coliO157 and six additional serogroups of Shiga toxin-producingE. coli(STEC) (O26, O45, O103, O111, O121, and O145) account for the majority of STEC infections in the United States. In this study, O serogroup-specific genes (wzxorwzy) were used to design loop-mediated isothermal amplification (LAMP) assays for the rapid and specific detection of these leading STEC serogroups. The assays were evaluated in pure culture and spiked food samples (ground beef, beef trim, lettuce, and spinach) and compared with real-time quantitative PCR (qPCR). No false-positive or false-negative results were observed among 120 bacterial strains used to evaluate assay specificity. The limits of detection of various STEC strains belonging to these target serogroups were approximately 1 to 20 CFU/reaction mixture in pure culture and 103to 104CFU/g in spiked food samples, which were comparable to those of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. In various beef and produce samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of respective STEC strains, the LAMP assays consistently achieved accurate detection after 6 to 8 h of enrichment. In conclusion, these newly developed LAMP assays may facilitate rapid and reliable detection of the seven major STEC serogroups in ground beef, beef trim, and produce during routine sample testing.

Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 55%

Rapid and Specific Detection of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157 in Ground Beef, Beef Trim, and Produce by Loop-Mediated Isothermal Amplification

Jiang

Yang

et al. 2012

Appl Environ Microbiol

“…Previously, LAMP assays have been developed for the detection of generic (22) and pathogenic E. coli, including enteroaggregative E. coli (44), enteroinvasive E. coli (37), enterotoxigenic E. coli (43), STEC (20,28,29), and more specifically, E. coli O157:H7 (40,46,47). With 35 min to 1 h of reaction time, these LAMP assays were capable of detecting between 0.7 and 100 CFU of E. coli per reaction, 10-to 100-fold more sensitive than conventional PCR (20,22,28,37,40,44,47).…”

Section: Discussionmentioning

confidence: 99%

“…With 35 min to 1 h of reaction time, these LAMP assays were capable of detecting between 0.7 and 100 CFU of E. coli per reaction, 10-to 100-fold more sensitive than conventional PCR (20,22,28,37,40,44,47). The three LAMP assays developed here fell within these detection ranges in terms of speed and sensitivity.…”

Section: Discussionmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification Assays for Detecting Shiga Toxin-Producing Escherichia coli in Ground Beef and Human Stools

Jiang

2012

J Clin Microbiol

Shiga toxin-producing Escherichia coli (STEC), encompassing E. coli O157 and non-O157 STEC, is a significant cause of foodborne illnesses and deaths in the United States and worldwide. Shiga toxins (encoded by stx) and intimin (encoded by eae) are important virulence factors for STEC strains linked to severe human illnesses such as hemorrhagic colitis and hemolytic-uremic syndrome. In this study, the stx 1 , stx 2 , and eae genes were chosen as targets to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, sensitive, and quantitative detection of STEC strains. The assay performances in pure culture and spiked ground beef and human stools were evaluated and compared with those of quantitative PCR (qPCR). No falsepositive or false-negative results were observed among 90 bacterial strains used to evaluate assay specificity. The limits of detection for seven STEC strains of various serogroups (O26, O45, O103, O111, O121, O145, and O157) were approximately 1 to 20 CFU/reaction in pure culture and 10 3 to 10 4 CFU/g in spiked ground beef, which were comparable to the results of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. When applied in ground beef samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of STEC cultures, the LAMP assays achieved accurate detection after 6 to 8 h enrichment. The assays also consistently detected STEC in human stool specimens spiked with 10 3 or 10 4 CFU/0.5 g stool after 4 h enrichment, while qPCR required 4 to 6 h. In conclusion, the LAMP assays developed in this study may facilitate rapid and reliable identification of STEC contaminations in high-risk food commodities and also facilitate prompt diagnosis of STEC infections in clinical laboratories.

“…Since it is isothermal, LAMP can be performed in much simpler instruments, such as a heater or water bath. To date, multiple LAMP assays targeting STEC Shiga toxin genes (stx 1 and stx 2 ) have been developed (20)(21)(22)(23)(24) and evaluated in food, primarily beef (25)(26)(27)(28), as have several others targeting the E. coli O157 rfbE gene (encoding perosamine synthetase) (23,24,(29)(30)(31). Very recently, we developed a suite of LAMP assays for STEC (targeting common virulence genes stx 1 , stx 2 , and eae) and the seven adulterant STEC serogroups (targeting the wzx or wzy gene on the respective O-antigen gene clusters) (32,33).…”

mentioning

confidence: 99%

Evaluation of a Loop-Mediated Isothermal Amplification Suite for the Rapid, Reliable, and Robust Detection of Shiga Toxin-Producing Escherichia coli in Produce

Yang

et al. 2014

Appl Environ Microbiol

c Shiga toxin-producing Escherichia coli (STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n ‫؍‬ 156) and various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4°C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 10 5 to 10 6 CFU per 25 g (i.e., 10 3 to 10 4 CFU per g) in produce, except for strains harboring the stx 2c , eae-␤, and eae-subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx 2 and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis.