Detection of Listeria monocytogenes in ready to eat

Detection of Listeria monocytogenes in ready to eat (RTE) samples of meat and chicken by loop-mediated isothermal amplification (LAMP) and PCR

Abstract
The prevalence and genetic diversity of Listeria monocytogenes in ready-to-eat (RTE) meat and chicken (white meat) was studied in retail marketing of different supermarkets and open-air markets in Gorgan city, Iran. A total of 60 RTE meat and chicken samples were purchased, and then, defined into two groups. 15% of collected samples (at retail level) were found to be contaminated by L. monocytogenes. Contamination of RTE chicken meat (20%) was higher than that of the RTE meat (10%). . The detection accuracy was 100%, which was higher compared to traditional culture method (15%). These results indicate a better detection ability of LAMP compared to the conventional PCR method. The contamination prevalence in open-air markets (54.5%) was higher than that of supermarkets (45.5%), which revealed the poor sanitary conditions of open-air markets. One important achievement of this research was that majority isolates (93.9%) from the positive samples belonged to serogroup ?of 1/2a, 1/2b, 3b (potential serotype of 1/2a or 1/2b), which is associated with the human listeriosis, and may cause the public health issues in Gorgan.

1. Introduction
L. monocytogenes, which is responsible for listeriosis, is a gram-positive and a foodborne bacteria, which can cause severe harms, or even, mortality, especially in immunocompromised persons,. According to FoodNet and the European Food Safety Agency (EFSA, 2013), L. monocytogenes infections are associated with fatality rate of approximately 12%, which is the highest amount among foodborne pathogens (EFSA, 2013).
L. monocytogenes lives in the soil and water, and can be found in infected domestic and wild animals (EFSA, 2013). about samples which reported to be taken under processing, the absence of L. monocytogenes in 25 g, was considered as a criteria ,applied for each sample. The samples of hard cheeses and fermented sausages, was exception, science these categories are assumed to be unable to support the growth of L. monocytogenes. For these samples, the limitation of ?100 CFU/g was applied in processing (EFSA, 2013). The human listeriosis is transmitted, through contaminated foods (Mead et al., 1999; WHO, 1988). Also L. monocytogenes are inactivated by pasteurization and cooking, but found mainly, in ready-to-eat (RTE) foods such as cheese, poultry products, RTE meat products, vegetables, seafood’s and salads (Nuvolosi et al., 2006). other ability of L. monocytogenes is to form biofilm, which can grow at freezing temperatures (2-4?), and tolerate high concentrations of NaCl. (Samelis et al., 2002; Gandhi ; Chikindas, 2007). Contamination of equipment in meat processing sites and food processing locations may be lead to L. monocytogenes contaminating (Loura, Almeida ; Almeida, 2005). L. monocytogenes strains are different in their epidemic potential and indisposing ability. Among 13 L. monocytogenes serotypes, serotypes of 4b, 1/2a and 1/2b are responsible for more than 95% of listeriosis (Kathariou, 2002). The human listeriosis caused by a small number of epidemic clones (ECs): ECI, EC Ia and ECII (within serotype 4b) and ECIII (serotype 1/2a) (Kathariou, 2002, 2003; Olsen et al., 2005).
A total of 1096 food samples were purchased from Tetouan, North-Western of Morocco, to examine the presence of Listeria Spp. 7.3 percent of the tested samples had been infected by Listeria spp., while L. monocytogenes was detected in only 16 samples (1.5% of total) (Amajoud et al, 2018).
The highest contamination in food samples, was observed in RTE fishery products (6.7%), while the contamination rate at the batch state, was 2.3%. In cheese processing, the highest contamination rate was related to unspecified cheeses: 2.1 % in single samples and 5.4 % in batches (EFSA, 2013). According to Kramarenko et al. 370 RTE food samples were collected at retail level, from which, 11% were found to be infected by L. monocytogenes. The contamination among RTE fish products (17%) was higher than that of in RTE meat products (6%) (Kramarenko., Et al 2016).
In recent years, as development of modern molecular diagnosis technologies, the PCR-based methods, with improved detection resolution and shortened analysis time, can be used to detect L. monocytogenes in food samples, include multiplex PCR (Jamali et al., 2015; Wieczorek ; Osek, 2017), immune-magnetic separation PCR (Ayaz, Ayaz, Kaplan, Dogru, ; Aksoy, 2009), real-time PCR (Traunsek et al., 2011).
Notomi et al. developed the loop-mediated isothermal amplification (LAMP) method, which is simple, fast and field-adaptable technique (Notomi et al., 2000). This method employs a set of specially designed primers, based on six or eight distinct regions of target DNA, as the entire process can continuously yields millions of long DNA concatamers under isothermal conditions in 1 h (Nagamine, Kuzuhara, ; Notomi, 2002; Notomi et al., 2000). this method is quite simple and easy to perform. It only requires a water bath or a heating tool, and a Bst DNA polymerase, for reaction progress, and amplifications can be visually determined by adding fluorescence intercalation dye or metal ion indicators, followed by observing color changes with eye or under ultraviolet (UV) (Tomita, Mori, Kanda, ; Notomi, 2008; Parida M et al, 2008). The main advantage of LAMP over PCR is that, it does not require expensive thermal cyclers as well, the amplification can be conducted in 1 h in a laboratory heating block or even a water bath. LAMP results can be observed by a visual turbidity, using a turbidometer, or by the adding fluorescent reagents, such as SYBR Green I (Iwamoto et al, 2003: Mori et al, 2001).
In the present study, the LAMP technique was used to study L. monocytogenes diversity in meat samples from retail markets in Gorgan, Iran. Subsequently, the LAMP was carried out using specific primers, which designed according to the actin polymerization gene (actA) of L. monocytogenes. The sensitivity, specificity, and reproducibility of this method were assessed, and its application in L. monocytogenes detecting, in real food samples, was also investigated.

2.1. sampling
A total of 60 samples were purchased from deli section of 12 supermarkets, and 29 open-air markets, from September to November 2017 in Gorgan city( in northern of Iran). The samples were divided into two groups. Those which analyzed in this study, were consisted chicken products (include wings, neck, thigh, chest), as well, ready-to-eat (RTE) products of meat. All these RTE products, were sampled in aseptic conditions, and then, were transported into a freezer, in the laboratory for 2h.

2.2. Isolation and numeration of L. monocytogenes, and genomic DNA extraction
L. monocytogenes was isolated according to the United States Department of Agriculture methods (MLG 8.10( as follow: 25g of each sample was incubated in 225mL Listeria enrichment medium at 30? for 24h, and then 0.1mLpre-enrichment medium was added into the culture medium, which was spread into CHROM agar Listeria, and then, incubated for 48h at 37?. All culture mediums were obtained from Food and Drug Administration in Iran. Five typical Listeria colonies, were selected from each selected agars, and then, were streaked into a new CHROM agar Listeria and incubated for 48h at 37?.
The confirmation tests were performed using pure culture obtained from BHI (Merck, Germany) agar. Isolated species were catalase and gram positive contain tumbling motility, which inoculated on 5% sheep blood agar (Merck, Germany) plates for determining the hemolytic reaction, ?-hemolysis for L. monocytogenes. For further confirmation, carbohydrate utilized along with rhamnose and xylose, in OF basal culture medium (pro media culture, Iran). For qualifying the consumption of mannitol, the inoculate tests were performed in mannitol salt agar medium (Merck, Germany) .

2.3. extraction of DNA from the isolated L. monocytogenes
In the following, the genomic DNAs were separated from 1 mL of tested bacterial culture mediums, using a “Bacterial Genomic DNA Purification Kit” (DENA Zist Asia, Iran), according to the manufacturer’s instructions. DNA samples were re-suspended in 50 mL ddH2O, and were stored at -20 C.

2.4. Primer designing for LAMP assay
The hlyA sequence of L. monocytogenes, was downloaded from NCBI database (accession number NC012488), and LAMP primer sets, were designed by Primer Explorer Version 4.0 (http://primerexplorer.jp/elamp4.0.0/index.html), according to the sequence. Finally, one primer set, was selected after checking the specificity by NCBI BLAST (Basic Local Alignment Search Tool), and was screened by Premier 5.0 (PREMIER Bio soft International, Palo Alto, CA), to analyze dimer and false priming formation. The sequences and locations of each primer are shown in Table 1.

2.5. LAMP reaction
The temperature and reactions time of LAMP processes were optimized by the using of L. monocytogenes strain of PTCC 1298. The final LAMP assay consisted of the following components: 0.2 µmol l-1 of each outer primer (F3 and B3), 1.6 µmol l-1 of each inner primer (FIP and BIP), and 0.8 µmol l-1 of each loop primer (LF and LB), 1.4 mM dNTPs mixture, 0.6 M betaine (Sigma, St. Louis, MO, USA), 6 mM MgSO4, 8 U Bst DNA polymerase (New England Biolabs, Beverly, MA,USA), 19 thermopol buffer, 2 µl template DNA, and 0.05 mmol l-1 calcein, and 0.6 mmol l-1 manganese ions, overally 25 µl. The mixture was incubated at 60°C with an amplification time of 60 min, followed by incubation at 80°C for 5 min, to terminate the elongation reaction for inactivating the Bst DNA polymerase. DNA-less Mixture was used as the negative control sample.

2.6. PCR assay
PCR reactions progress in optimized conditions which contain the components of L. monocytogenes strain PTCC 1298. The PCR investigation was performed as follow: PCR was carried out in 25 µl volume of reaction mixture containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 0.2 lM of each F3 and B3 primer, 0.2 mM of each dNTP, 1 µl DNA template, and 0.5 units of Taq DNA polymerase (Ex Taq; Takara). The experiment was performed in 35 cycles of denaturation, annealing, and extension (30 s at 95 ?C, 30 s at 56 ?C, and 1 min at 72 ?C, respectively) totally 10 min for extension step at 72?C. PCR products, were electrophoresed to verify the expected 346 bp bands.

2.7. Analysis of the LAMP Products
The initial amplicon and digesting of products, was processed in electrophoresis tools for 50 min with 2% agarose gels followed by visualizing on UV trans illuminator, at 254 nm, which consequentially, the specific ladder of multiple bands was observed. the specificity of amplification was further confirmed by restriction enzyme which digest the LAMP products.

3. RESULT and DISCUSSION
The overall prevalence of L. monocytogenes in RTE meat and chicken products, is shown in table 2. The measure of L. monocytogenes contaminated RTE chicken products (20%) was significantly higher than that of RTE meat products (10%). L. monocytogenes comprise 13 serotypes (1/2a,1/2b,1/2c, 3a, 3b, 3c, 4a, 4b, 4ab, 4c, 4d, 4e, and 7), but 95% of the strains extracted from foods and patients, belonged to serotypes of 1/2a, 1/2b, and 4b (Farber & Peterkin, 1991; Graves, Swaminathan & Hunter, 2007). The results of this probe was in agreement with the our previous research (Kramarenko et al, 2013; Zhang et al., 2007; Parihar et al., 2008). The predominant serogroup was 1/2b (potentially serotype 1/2b) (61.5%), whereas in some other studies it was 1/2a, suggesting the rank of each serotype, to may be different in other studies performed in various locations.
The prevalence of L. monocytogenes in RTE meat products is generally low, but controversially, in a recent study performed in china, a total of 628 ready-to-eat (RTE) meat products had been investigated, 33 sample (5.3% of total samples) were contaminated, which confirmed by the bacteriological method and PCR, include 7.2% (17/236) of sauce pickled products, 4.2% (11/260) of cured products and 5.6% (5/90) of smoked and roasted products (Wang., Et al 2014). In a study in Greek, 28% of tested RTE meat products, had been contaminated by L. monocytogenes (Manios et al. 2014). Also, according to the Belgium, L. monocytogenes was observed in 27.8% (25/90) of smoked fish samples (Uyttendaele et al., 2009). In recent study All the RTE meat products of Estonian origin were compatible with EU food safety criteria, which was higher compared to the of RTE fish (1.6% ) and of RTE meat products (0.7% ) (Kramarenko et al, 2016). Although in previous Baltic studies, cold-smoked pork and beef products, had been found to have a high L. monocytogenes prevalence (Berzins, Horman, Lunden, & Korkeala, 2007; Berzins, Terentjeva, & Korkeala, 2009). according study of Giza Governorate, Egypt, a total of 150 progressed meat samples were collected. The identification of phenotypic and genotypic of Listeria monocytogenes was performed by applying of PCR incorporating listeriolysin O virulence gene hlyA, followed by DNA sequence analysis. L. monocytogenes was detected in 4% of each beef burger, minced meat, and luncheon samples (Mohamed et al, 2016).
Similarly, the European baseline survey, which carried out in 2010 and 2011, qualified the EU wide prevalence of L. monocytogenes as the highest one among the chicken products (10%) and the lower one among the meat products 2.1%, at the end of products’ life time (EFSA, 2013). Similarly, the prevalence of RTE chicken has been reported in different studies, performed in Thailand. The examination of 200 raw chicken meat samples, showed that 51 of samples (25.5%) was infected by L. monocytogenes. In Brazil a total of 920 breast and 774 thigh samples, were analyzed, and L. monocytogenes was detected in 8.64 and 44.19% of the samples, respectively (Schäfer et al, 2017). The purpose of the study in Amman, Jordan was to determine the prevalence of Listeria spp. The results showed that In raw chicken and ready-to-eat (RTE) chicken products, among of 280 samples, 141 sample (50%) were found to be contaminated by Listeria spp. L. monocytogenes (18.2%), Listeria ivanovi (26.1%), Listeria grayi (3.5%), Listeria seeligeri (1.8), Listeria welshimeri (0.7%) (Osaili et al, 2011). The prevalence of L. monocytogenes in ready-to-eat chicken meat samples, sold in retail markets in Turkey, was investigated, and Listeria species were qualified in 12 (24%) examined samples. Among the extracted Listeria species, the number of L. monocytogenes, L. innocua, and L. welshimeri were, 9, 2, and 1, respectively (Seçil, 2017).
As shown in table 3, sensitivity of the LAMP and PCR investigation for L. monocytogenes strain with pure cultures, were demonstrated. To demonstrate the utility of LAMP as a probe for L. monocytogenes in food, 60 samples were analyzed by LAMP, conventional PCR, and culture–biotechnical methods. The results are summarized in table 3 and Fig1,2. In the case of RTE chicken and meat samples, 9 (15%) and 7 (11.6%) samples were identified by LAMP and PCR to contaminated by L. monocytogenes (Table 3), respectively. The detection accuracy obtained by 100%, which compared to the traditional culture method (15%), was better. These results suggest a higher detection ability by the LAMP assay compared to the conventional PCR.

4. Conclusion
In recent years, several outbreaks of L. monocytogenes, have been connected to the RTE products (http://www.cdc.gov/listeria/outbreaks/). These products are regarded to be at a higher risk for L. monocytogenes contamination, than that of in other foods, but little information is available about it in Iran.
In our study, the overall prevalence of L. monocytogenes in RTE products was reported as 15%. In another reports(Osaili, Alaboudi ;Nesiar, 2011; Fallah et al.,2011; Yu ; Jiang, 2014) the L. monocytogenes was extracted from RTE meats in different measures as 30%, 11.4% and 6.3% in Jordan, Iran and China respectively. However, in contrast to other studies (Uyttendaele et al., 1999; Baek et al., 2000; Mena et al., 2004; Vitas et al., 2004; Stonsaovapak ; Boonyaratanakornkit, 2010; Yan et al., 2010), the contamination of RTE foods was found to be at a lower rate. The detection accuracy was obtained as100%, which was higher compared to the traditional culture method (15%). These results suggest a higher detection ability gained by LAMP assay compared to conventional PCR.
The present research, also, indicated that sauce pickled chickens and grilled meat products can be the potential sources of L. monocytogenes (table 2). The contamination level in open-air markets (54.5%) was higher than that of in supermarkets (45.5%), which revealed the worse sanitary condition of open-air markets up to date. One of important points revealed in this study, was that, the majority difected extracts (93.9%) belonged to the serogroup 1/2a, 1/2b, 3b (mainly serotype of 1/2a or 1/2b), which is associated with human listeriosis, and may cause a public health issue in Gorgan.