A one-step rapid screening test ofListeria monocytogenesin food samples using a real-time loop-mediated isothermal amplification turbidity assay

Abstract

A rapid and specific, hly-based, loop-mediated isothermal amplification (LAMP) was applied for the detection of Listeria monocytogenes in food and food products, using a real-time turbidimeter platform (LAMP-turbidity). The principle behind this method relies on an increase in a DNA yield, which correlates with the production of magnesium pyrophosphate, and the results can be determined via an amplification curve within 1 h. The specificity test revealed that L. monocytogenes (DMST 17303) was observed from 34.1 to 38.3 min, while thirty strains of non-L. monocytogenes demonstrated no crossreactions. The limits of detection for purified genomic DNA and pure culture were 800 pg mL À1 and 2.82Â 10 3 CFU mL À1 , respectively. Investigation on 200 raw chicken meat samples indicated that the specificity, sensitivity, and accuracy of LAMP-turbidity were 100%, 62.75%, and 90.50%, respectively.These data suggest that an hly-based, real-time, quantitative LAMP-turbidity assay can be an applicable tool for the epidemiological screening of L. monocytogenes in food and food products.

Rapid Colorimetric Assay for Detection of Listeria monocytogenes in Food Samples Using LAMP Formation of DNA Concatemers and Gold Nanoparticle-DNA Probe Complex

“…The LAMP reaction is detected by measuring the fluorescent intensity of the indicated reagents (Fischbach et al, 2015 ) or the turbidity of the DNA polymerization by-products (magnesium pyrophosphate; Mori et al, 2001 ; Jayawardena et al, 2007 ). These methods are capable of obtaining real time signals, from which the amount of the DNA template can be quantified (Liu et al, 2017 ; Wachiralurpan et al, 2017b ). The visual detection of turbidity by the naked eye was achieved when a fluorescent dye such as SYBR green was added to the solution after the LAMP reaction (Mashooq et al, 2016 ).…”

In-Situ Monitoring of Real-Time Loop-Mediated Isothermal Amplification with QCM: Detecting Listeria monocytogenes

“…To assess this hypothesis, we tested five different initial concentrations of L. monocytogenes DMST 17303 gDNA samples. Figure 4 shows the results: when injecting a LAMP mixture containing approximately 0.8 ng of L. monocytogenes gDNA, starting amplification led to a signal onset after 27 min It is worth noting that this clearly showcases an added advantage of the QCM-based assay: turbidity measurements at 650 nm showed a signal only after 46 min [ 21 ]. Switching to measuring on the QCM in situ hence improves sensitivity by decreasing the measuring time.…”

“…Modified QCM were placed in a custom-made PDMS cell for measurements. The temperature was kept at 60 °C by a thermostat (Thermo Haake ® DC30, Gebrüder HAAKE GmbH, Karlsruhe, Germany), which turned out the optimal temperature for LAMP amplification [ 21 ] when developing a colorimetric test for a different gene of L. monocytogenes . This cell was then connected to a custom-made oscillator circuit to operate the device.…”

“…The LAMP mixture used here is the result of previously published optimization [ 21 ], which revealed that one can use the turbidity signal at 650 nm as a measure of determining how far the LAMP process has proceeded. It makes use of the increasing amount of white magnesium pyrophosphate precipitate in the solution.…”

Ultrasensitive detection of Listeria monocytogenes using solid‐state electrochemiluminescence biosensing based on the quenching effect of ferrocene on ruthenium pyridine

“…Traditional microbiological and biochemical assays for the detection of Listeria monocytogenes rely on preliminary culture, followed by biochemical and serological identification, taking at least 3 days. More recently, traditional methods are gradually being replaced by those that are more convenient and simple, such as immunological (Zhang, He, Liu, Peng, & Li, 2014), molecular biological (Wachiralurpan, 2017; Yan Wang, 2018), or electrochemical biosensor method techniques (Suh et al, 2018; Yang, Zhou, Zhu, & Xing, 2017). Among the many detection methods, the design of ECL biosensors, including solution‐state and solid‐state ECL systems has recently become an active research focus.…”