Visual Detection of Enterohemorrhagic Escherichia coli O157:H7 Using Loop-Mediated Isothermal Amplification

Abstract

IntroductionEscherichia coli O157:H7, an important foodborne pathogen, can cause serious renal damage, which can also lead to mortality. Since a rapid and sensitive method is needed to identify this pathogenic agent, we evaluated Loop-Mediated Isothermal Amplification Assay (LAMP) to detect Escherichia coli O157:H7.MethodsWe used six primers that specifically identified the rfbE gene. To examine the sensitivity of the method, different dilutions were subjected to the LAMP reaction. Other bacterial strains also were investigated to determine the specificity of the test. The turbidity of the amplified products was assayed by visual detection. The amplified products were detected by addition of SYBR Green II to the reaction tubes.ResultsAmplification products were observed as a ladder-like pattern on the agarose gel. A white turbidity emerged in the positive tubes. Under UV light, the positive samples were green, whereas the negative samples were orange. The detection limit of the LAMP was 78 pg/tube, and this indicated that it was 100 times more sensitive than PCR for the detection of EHEC. No LAMP products were detected when template DNA of non-EHEC strains were used, suggesting high specificity of the LAMP assay.ConclusionThe results indicated that the LAMP assay is a valuable diagnostic assay to identify EHEC O157:H7. In addition, the simplicity, sensitivity, specificity, and rapidity of this assay make it a useful method to diagnose pathogens in primary labs without any need for expensive equipment or specialized techniques.

Rapid Detection of Escherichia coli O157:H7 by Loop-Mediated Isothermal Amplification Coupled with a Lateral Flow Assay Targeting the z3276 Genetic Marker

“…Second, as shown by the results in Fig. 3, this assay had higher sensitivity than previously reported LAMP assays (Ranjbar et al 2016;Zhu et al 2009). For example, the LAMP assay targeting the rfbE gene developed by Zhu and colleagues allowed for the positive detection of E. coli O157:H7 with a sensitivity of 26 and 10 CFU/reaction (Zhu et al 2009), whereas the z3276-LAMP-LF detection limit was 1.3 CFU/mL.…”

“…Several LAMP methodologies have previously been reported for the detection of E. coli O157:H7 that involve amplifying various target genes, including O-serogroup specific genes (rfbE, wzx, wzy), Shiga toxin genes (stx1 and stx2) (Ranjbar et al 2016;Dong et al2014;Wang et al 2012), and E. coli attaching and effacing gene (eae) (Zhang et al 2013); however, most of these genes are not unique genetic markers for this serotype (Ravan and Amandadi 2015). The inability of these genes to accurately differentiate E. coli O157:H7 from other E. coli serotypes prompted the investigation of novel genetic markers.…”

Loop‐Mediated Isothermal Amplification (LAMP): A Rapid and Sensitive Tool for Quality Assessment of Meat Products

“…Wang and others () also used the EMA–LAMP method for the detection of Vibrio parahaemolyticus in VBNC and dead state. Ranjbar and others () used SYBR Green II with the LAMP method for visual detection of EHEC O157:H7. The positive samples showed green color whereas negative samples were orange in color under UV light.…”

Real‐time loop‐mediated isothermal amplification assays combined with ethidium monoazide bromide and bentonite coated activated carbon for rapid and sensitive detection of viable Escherichia coli O157:H7 from milk without enrichment

“…Recently, a LAMP assay was invented which is time-saving, simple to operate, and appropriate in primary clinics or field labs (Notomi et al, 2000). Meanwhile, it can amplify without the process of denaturation and renaturation of nucleic acid under isothermal temperature with 2-3 pairs of primer for more than 10 min and result in 10 9 -10 10 copies of target DNA within 1 hr (Kokkinos, Ziros, Bellou, & Vantarakis, 2014;Ranjbar et al, 2016).…”

“…Previous studies (Luan & Levin, ; Wu et al, ) have shown that the recovery of foodborne pathogens was significantly improved after bentonite coated activated carbon (BCAC) treatment. Currently, the majority of quality control systems available for the supervision of milk in the food industry use an initial enrichment followed by a detection system such as PCR or LAMP assay (Kokkinos et al, ; Ranjbar et al, ). Such methodology timely precludes the presence of pathogen before the product is shipped.…”