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

Abstract

<jats:title>ABSTRACT</jats:title><jats:p><jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>O157 and six additional serogroups of Shiga toxin-producing<jats:named-content content-type="genus-species">E. coli</jats:named-content>(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 (<jats:italic>wzx</jats:italic>or<jats:italic>wzy</jats:italic>) 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 10<jats:sup>3</jats:sup>to 10<jats:sup>4</jats:sup>CFU/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.</jats:p>

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

“…Among them, seven STEC strains (underlined in Table 1), one from each of the seven adulterant serogroups, were used for sensitivity testing and spiked-produce experiments. All strains were cultured as described previously (33).…”

“…A LAMP suite of 10 assays recently developed by our research group (32,33) was evaluated. The first three assays targeted common STEC virulence genes (stx 1 , stx 2 , and eae), while the remaining seven each targeted a gene (wzx or wzy) on the O-antigen gene clusters of the seven adulterant STEC serogroups.…”

“…The assays were performed as described previously (32,33). Briefly, the LAMP reagent mix (25 l) contained 1ϫ ThermoPol reaction buffer (New England BioLabs, Ipswich, MA), 6 mM MgSO 4 , 1.2 mM each deoxynucleoside triphosphate (dNTP), 0.1 M each outer primer (Integrated DNA Technologies, Coralville, IA), 1.8 M each inner primer, 1 M each loop primer, 10 U of Bst DNA polymerase (New England BioLabs), and 2 l of template DNA.…”

“…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). Although it has been reported to be rapid, specific, and sensitive, the LAMP suite has not been evaluated using a large number of STEC strains harboring various stx and eae subtypes or tested with a variety of produce items using conditions mimicking real-world contamination events (e.g., low-level surface inoculation, cold storage).…”

Real-time DNA Amplification and Detection System Based on a CMOS Image Sensor

“…In addition, LAMP techniques could be integrated to a microfluidic chip, 12 lab-on-chip, 13 thermal platform, and various detectors, including electrophoresis, 14 turbidimetric methods 15 and electrochemical sensors real-time disposable LAMP system for gene amplification and detection using a saliva sample within 30 min. The study demonstrated an ion-sensitive field-effect transistor integrated to a complementary metal oxide semiconductor (CMOS) sensor for DNA amplification and detection by measuring the hydrogen ion release.…”