DEVELOPMENT AND EVALUATION OF A LOOP‐MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) METHOD FOR DETECTING LISTERIA MONOCYTOGENES IN RAW MILK

Abstract

The performance of a Loop‐mediated isothermal amplification (LAMP) assay for detecting food‐borne pathogen Listeria monocytogenes was presented in this paper. Three pairs of primers were specially designed for recognizing eight distinct sequences of iap gene (P60 extracellular protein, invasion associated protein IAP). Time and temperature conditions for amplification of L. monocytogenes were optimized to be 40 min at 63C. Detection limit level for artificially contaminated raw milk samples by the LAMP assay was 186 cfu/mL corresponding to 8–10 cells per reaction tube, while the detection level by conventional PCR was 1.86 × 105 cfu/mL. Data on natural raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 91.67% concordance with the ISO 10560 reference method for the samples without enrichment and 100% concordance after enrichment. PRACTICAL APPLICATION The LAMP method in this paper provided a powerful tool with the specificity, sensitivity and rapidity for detection of Listeria monocytogenes in raw milk.

Rapid Detection of Listeria monocytogenes in Raw Milk with Loop‐Mediated Isothermal Amplification and Chemosensor

“…2008; Okamura and others. 2008; Wang and others 2008; Wang and others 2009), but the detection limit of the PCR method was higher than that reported for L. monocytogenes (Rossmanith and others 2006), which may be due to the wide range of inhibitors in template DNA extracted from artificially contaminated raw milk (Kaneko and others 2007). …”

“…In LAMP, a large amount of DNA was synthesized, yielding a large amount of the pyrophosphate ion as a by‐product. The reaction of pyrophosphate ion with rhodamine‐based dual chemosensor and Cu 2+ results in the disappearance of the red colour, which enabled visual discrimination of results, and gives the rhodamine‐based dual chemosensor method advantages over agarose gel electrophoresis (Wang and others 2010), turbidity (Seki and others 2005), SYBR GREEBI (Bipin and others 2007), calcein (Tomita and others 2008), and lateral flow dipstick (Teeranart and others 2010). Agarose gel electrophoresis has a carcinogenic hazard and can be confused easily by cross contamination; determination of turbidity needs expensive instrumentation – Loopamp real‐time turbidimeter; SYBR GREEBI cannot discriminate between amplified DNA and primer dimers, which frequently resulted in a false positive result (Daniel and others 2007); visual analysis with calcein was performed under a fluorescent lamp, and the lateral flow dipstick is based on both labeled primer and probe, which increases test cost and perplexed detection process.…”

A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Listeria Monocytogenes

“…In terms of reaction time, the iap ‐based LAMP assay took 40 min, whereas the hlyA ‐based LAMP assay only took 20 min, half of the time needed for the former. The detection limits of the hlyA ‐based LAMP assay are up to 1.74 and 20.4 cfu/tube in pure culture and contaminated chicken samples, respectively, which are compatible with those of the iap ‐based LAMP assay at 8–10 cells per tube for artificially contaminated raw milk (Wang et al . 2010).…”

“…Our data regarding the specificity of the hlyA-based LAMP assay clearly indicated its potential application in foodborne pathogen detection. Additionally, Wang et al (2010) established a LAMP assay to detect L. monocytogenes in raw milk by utilizing the invasion associated protein (iap) gene (P60 extracellular protein) as the target gene. In terms of reaction time, the iap-based LAMP assay took 40 min, whereas the hlyA-based LAMP assay only took 20 min, half of the time needed for the former.…”

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

“…Although, there were many rapid detection methods designed based on PCR and LAMP assays for foodborne pathogen, which could be limited in sensitivity by DNA amplification inhibitors and caused false-negative results (Wang, Huo, Ren, & Li, 2010;Wu, Chen, & Levin, 2015). In milk, components such as Ca 2+ , proteinase, fats, and milk proteins may block DNA and shield it from access by polymerase (Soejima et al, 2008;Wilson, 1997).…”