Development of a loop‐mediated isothermal amplification assay targeting lmo 0753 gene for detection of Listeria monocytogenes in wastewater

Abstract

Contaminated wastewater plays an important role in the transmission of Listeria monocytogenes in the environment. In this study, a loop‐mediated isothermal amplification (LAMP) assay for sensitive detection of L. monocytogenes in wastewater from treatment plants was developed, validated and compared to conventional PCR. The lmo0753 gene which codes for a Crp/Fnr family transcription factor, was targeted to design four specific primers to detect L. monocytogenes in 60 min at 63°C in a water bath. Amplification products were visualized by agarose gel electrophoresis. The detection limit of the LAMP assay was 65 fg µl−1 of DNA and 38 CFU per ml. Conventional PCR was 10 times less sensitive than LAMP assay with primers targeting the HlyA gene. A total of 70 crude wastewater samples collected at different treatment stages (aeration tank, pre chlorination and post chlorination), were tested directly by LAMP and PCR. Samples from aeration and pre‐chlorination stages tested positive with LAMP and culture method but not with conventional PCR. LAMP assay was tolerant to inhibitors present in wastewater and circumvented the need for isolation of pure DNA for detection. Both LAMP assay and culture method failed to detect L. monocytogenes in post‐chlorinated wastewater, confirming the efficiency of the treatment process in the removal of L. monocytogenes. Significance and Impact of the Study Treated wastewater effluent contains Listeria monocytogenes which survives conventional wastewater treatment processes and can re‐enter human food chain, thus it is imperative to detect L. monocytogenes using a rapid and an inexpensive method. To the best of our knowledge, this is the first report of a loop‐mediated isothermal amplification (LAMP) assay, targeting the lmo0753 gene for detection of L. monocytogenes in wastewater from treatment plants. The LAMP assay detects L. monocytogenes in 60 min at 63°C in a water bath. LAMP does not require isolation of pure genomic DNA hence it is a user friendly method for L. monocytogenes detection.

Listeria monocytogenes in foods—From culture identification to whole‐genome characteristics

“…This gene was amplified during the identification of L. monocytogenes in chicken meat (Tang et al, 2011), raw meat, vegetables, seafood, different kinds of ready-to-eat foods of plant and meat origin (deli foods) (Shan et al, 2012), and dairy products (Tirloni et al, 2017). There are also other virulence genes such as pfrA, iap, lmo2234, lmo0737, which were successfully used for detection of L. monocytogenes with the LAMP approach (Costa et al, 2014;Nathaniel et al, 2019;Wang et al, 2015;Wu et al, 2014). A variant of conventional LAMP assay described by Wu et al (2014) contained primers specific for the hlyA and iap genes of L. monocytogenes.…”

Molecular Methods for Pathogenic Bacteria Detection and Recent Advances in Wastewater Analysis

“…The LoD was 65 fg/µL of DNA and 38 CFU/mL, which was 10 times more sensitive than conventional PCR with primers targeting the HlyA gene. However, in the application to wastewater, a pre-culture procedure at 37 • C for 48 h was required [179]. An SA23 probe targeting Salmonella specifically by FISH has been developed by Santiago et al (2008).…”

Direct Recombinase Polymerase Amplification Assay for Accurate and Rapid Detection of Listeria Monocytogenes in Food

“…Recently, isothermal amplification approaches based on conventional PCR assay such as loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), polymerase spiral reaction (PSR) and recombinase polymerase amplification (RPA) have been widely used in analyzing the foodborne organism pathogens ( Liu et al, 2015). LAMP, CPA and PSR assays were previously employed to detect L. monocytogenes (Wang et al, 2014;Du et al, 2018;Nathaniel et al, 2019). All of these methods were shown to have high sensitivity and specificity with the ideal limit of detection (LOD).…”