LAMP-based system provides rapid and reliable results for the detection L. monocytogenes in raw meat and meat products and can be applicable in meat industry. Sensitivity and specificity shold be more than 90%. Kappa-value should be more than 0.85.

L. monocytogenes ATCC 35152 NCTC 7973 and ATCC 13932 serovars 4b (from American Type Culture Collection (Manassas, VA, USA), were activated in 10 mL of tryptone soya broth (TSB, Oxoid, England) for 24 h at 37 °C. The cultures were centrifuged (Eppеndorf, Germany) at 3000 g for 10 min, washed twice with 0.1% (w/v) peptone water (Oxoid, England), and resuspended in 1 mL of 0.1% (w/v) peptone water (Oxoid, England), and then mixed (1:1, v/v) to prepare a 2-strain cocktail (101 and 102 CFU.mL-1). Before inoculation, the counts of prepared 2-strain cocktail of L. monocytogenes diluted in 0.1% (w/v) PW (Oxoid, England) were enumerated by spread plating an aliquot of 100 μL on tryptone soya agar (TSA, Oxoid, England) in duplicate and incubating TSA plates at 37 °C for 48 h to estimate the inoculum levels.

There were two inoculation levels for matrix: a high inoculation level of approximately 100 CFU._g-1 and a low inoculation level of approximately 10 CFU._g-1. Also was used uninoculated samples as negatives controls.

The detection of L. monocytogenes cells by the commercial LAMP-based kit (3M Molecular Detection Assay Listeria monocytogenes; 3M) was performed according to the manufacturer’s manual. Briefly, 25 g of sample were mixed with 225 ml Demi-Fraser broth (3M, USA). Then 20 μL of UVM enrichment was added to a tube with lysis solution. The mixture was warmed in a heat block (Germany, IKA) at 100 °C for 15 min, followed by immediate cooling at room temperature in a chilling block (3M, USA) for 10 min. After mixing by inversion, 20 μL of this lysate was mixed with the pellet in the reagent tube from the assay kit. The reagent tube was placed in a molecular detection system (3M, U.S.A.) for the detection of L. monocytogenes cells via isothermal amplification and bioluminescence for 75 min. All analyses included negative and reagent controls to validate the performance of the molecular detection system.

The samples were examined for the presence of L. monocytogenes bacteria in accordance with GOST 32031-2012. 25 g of the meat was homogenized in 225 cm3 of Demi-Fraser broth (Merck, Germany) and incubated at 30 °C for 24 hours. Then 0.1 cm3 the enriched culture was added to 10 cm3 of Fraser's broth (Merck, Germany) and cultured at 37 °C for 48 hours. From each broth after the end of incubation with a 3 mm loop, the enriched material was streaked onto a chromogenic agar for Listeria (Agar Listeria according to Ottaviani and Agost (Merck, Germany)) and selective nutrient agar for Listeria PAL (FBUN GNC PMB, Russia) and incubated at 37 °C within 24 – 48 hours. On chromogenic agar for Listeria, L. monocytogenes grows in the form of blue colonies with an area around; on PAL, brown colonies with black halos. Colonies typical of the genus Listeria and L. monocytogenes were seeded on tryptone soya agar with yeast extract (TSAYE) and incubated at 37 °C for 18 – 24 hours, which was then confirmed using biochemical tests (Oxoid, England).

Sensitivity and specificity of the commercial LAMP-based kit for the detection of L. monocytogenes were defined as the number of samples truly positive (T_Pos) and truly negative (Tneg), respectively, compared with the GOST method. The sensitivity, specificity, and accuracy of the commercial LAMP based kit were calculated as follows:

Sensitivity = [T_pos / (T_pos + F_pos)]

Specificity = [T_neg / (T_neg + F_neg)]

where T_Pos and TNeg are the number of positive and negative samples, respectively, confirmed by both the GOST and commercial LAMP-based kit, and Fpos and Fneg are the number of positive and negative samples, respectively, confirmed by the commercial LAMP-based kit. Kappa value of concordance, describing the statistical agreement between the two detection methods was calculated, as described. Kappa values were classified as follows: 0.01 indicated no concordance; 0.1 to 0.4 indicated weak concordance; 0.41 to 0.60 indicated clear concordance; 0.61 to 0.80 indicated strong concordance; and 0.81 to 1.00 indicated nearly complete agreement.

A chi-square test (AOAC, Official Methods of Analysis Program Manual) for significant difference was used to determine whether the proportion of positive samples was different between the 3M MDS and the GOST-method.

At the inoculum levels of 101 and 102 CFU._g-1 both methods abled the detection of L. monocytogenes in all samples (Table 1), resulting in 100% specificity and sensitivity (kappa value 1). At the inoculum level of 100 CFU._g-1 (not uninoculated L. monocytogenes), both methods were unable to detect L. monocytogenes. False negative results were not obtained.

The results samples show high specificity of the LAMP- method (not less than 90%) (Table 2). The 3M MDS result and culture-based detection (GOST 32031-2012 method) did not differ significantly (p >0.05) regarding the number of positive samples.

In similar studies, the high specificity and sensitivity of the method on artificially infected matrices has also been proven. L. monocytogenes was detected in 11 samples of pork by LAMP – method and in 10 samples by GOST 32031. One sample was not confirmed according to the reference method and was identified as false-positive (Fpos). The sensitivity of the method in the study of pork samples was 90% (Kappa value 0.93), specificity – 100%.

In another study the LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity, and accuracy were 100, 90.20, and 97.50%, respectively (Wachiralurpan et al., 2018).

Also, in the study of 32 samples of beef ready-to-cook products, 1 false positive result was found by the LAMP method. The sensitivity and specificity of the method were 88% and 100%, respectively, with a Kappa-value of 0.92.

In this case a false positive result could be caused by the DNA amplification on injured or sub lethally cells that cannot be detected by ISO (Lim et al., 2015). Another explanation for false positive results is the using of 4 to 6 primers with a much higher concentration in the LAMP method than in the classical methods based on PCR. In terms of efficiency, the PCR and real-time PCR assays could detect L. monocytogenes based on the listeriolysin O gene (hly) with a detection limit of 8 – 10 CFU (Rip and Gouws, 2009). However, these assays required sophisticated equipment and post-amplification manipulations that took more time to obtain results (Gianfranceschi et al., 2014).

This could lead to an increase in the possibility of non-specific amplification caused by forming primer dimers (Wang et al., 2015). 23 samples of beef were analysed. 5 positive and 18 negative results were detected and confirmed by both research methods (GOST and LAMP). However, 1 sample was false negative by LAMP-based method compared with GOST 32031-2012. The sensitivity of the method in the study of beef in this case was 100% (Kappa value 0.88), and the specificity – 94%. In other studies, false negative results were also obtained (Lim et al., 2015). These authors showed that 1 naturally contaminated sample of duck wings was presented as false-negative. The validation study also showed 91% sensitivity and 95% specificity, Kappa-value 1.

In the study of 19 ready-to-cook pork samples and 17 meat ready-to-eat products, no significant (p >0.05) differences in the results obtained by the LAMP and GOST 32031-2012 methods were found. The sensitivity and specificity of both methods was 100% with Kappa-value 1. Such a convergence of the two methods can be associated with an enough viable cells of L. monocytogenes in the sample to identify them.

Several false-positive and false-negative results were obtained at low levels of inoculum (10₁ CFU/10 cm₂), for the LAMP method have been reported (Mikš-Krajnik et al., 2015). It also reports at a 102 CFU/100 cm₂ microbial cell level, both methods were suitable for detection of L. monocytogenes and had 100% specificity and sensitivity.

In another study the LAMP method was employed to test 94 retail food samples effectively. Sensitivity in detection of L. monocytogenes by the LAMP was higher than that of PCR and none of the conventional method positive samples was missed by the LAMP method (Shan et al., 2012).

Listeriosis outbreaks were seen in many countries including Japan, the United States and countries of Europe (EFSA, 2011; Miya et al., 2015; Self et al., 2019). Human infections caused by L. monocytogenes have become a global health concern. The presence of L. monocytogenes in processing environment at slaughterhouses, deli meat factories or in retail may be a reason of cross-contamination. Listeria monocytogenes can contaminate various foods via food processing environments and contamination of raw materials. Hence, there is a necessity a variety of methods for rapid detection of foodborne pathogens as it is required in many food analyses.

In this study rapid LAMP method of L. monocytogenes detection was performed. Despite the having of false-negative and false-positive results, LAMP-based method was effective and easier to perform than some of standardized assays and has the advantage to reduce analysis time (less 2 days comparing with 5 days GOST method). Furthermore, this technique is 10 times more sensitive than the conventional PCR assay as reported (Wachiralurpan et al., 2017). The occurrence of false-positive results can be reduced by preventing cross-contamination, high humidity and temperature when working with reaction mixture tubes (Bird et. al.,2013, Wang et al., 2015).