PCR use for rapid identification of Listeria monocytogenes

The present research was conducted to check the presence of Listeria spp. in some meat and chicken products purchased from retail supermarkets in Assiut (Egypt). A total of 100 samples including 25 samples each of minced frozen beef, luncheon, frozen chicken legs and frozen chicken breast fillets were collected over a 7-month period between January and July 2009 and analyzed for the presence of Listeria spp. In addition, 28 stool cultures examined for Listeria spp. from hospitalized children resident in Assiut Pediatric University Hospital with diarrhea or fever. Out of the total 100 meat samples examined, Listeria spp. were detected in 8 (32%) of minced frozen beef, 8 (32%) of luncheon, 13 (52%) of frozen chicken leg and 14 (56%) of frozen chicken fillet samples analyzed, respectively. Regarding the examined 28 stool cultures from hospitalized children with underlying disease in Assiut Univ. hospital, 2 (7.14%) were found positive for Listeria spp. For 
identification of L. monocytogenes using polymerase chain reaction (PCR), two primers were selected to detect 217-pb fragment ofthe prfA (transcriptional activator of the virulence factor) gene for L. monocytogenes. 13 selected Listeria isolates displayed beta-haemolysis on sheep blood agar and positive CAMP test were further identified using PCR. PCR results showed that L. monocytogenes were confirmed in one of minced imported frozen meat examined, two of luncheon samples and two of frozen chicken legs with the total incidence of 5 isolates (5%) from the total 100 examined food samples. This suggests the presence of a significant public health hazard linked to the consumption of these meat and chicken products sold in Assiut city contaminated with L. monocytogenes. The public health significance of these pathogens as well as recommended sanitary measures was discussed.


Introduction
and L. monocytogenes are pathogenic in mice, but only L. monocytogenes is consistently associated with The marked increased of contamination in food human illness (Seafood Network Information Center, industry especially meat and chicken products by 2007).pathogenic bacteria has raised a great concern of the Listeria spp.has been isolated from poultry, red public.Listeria spp.especially L. monocytogenes has meat and meat products in many countries around the been associated with a wide variety of food sources world such as Yugoslavia (Buncic, 1991), Belgium particularly meat and chicken (Endang et al., 2003).
(Uyttendaele et al., 1999), New Zealand (Hudson et al., Listeria spp. is ubiquitous bacteria widely distributed in 1992), Australia (Ibrahim and Mac Rae, 1991), and the natural environment.The ubiquitous character of Japan (Ryu et al., 1992), although these foods have not the bacteria inevitably results in contamination of been associated with documented outbreaks of human numerous food products (Farber and Peterkin, 1991).
listeriosis.The detection of Listeria spp. in meat is of The genus Listeria includes 6 different species (L.monocytogenes, L. ivanovii, L. innocua, L. particular concern in terms of consumer safety, as welshimeri, L. seegligeri and L. grayi L. ivanovii these organisms are capable of growing on both raw ).Both and cooked meat at refrigeration temperatures (Walker detection of food borne pathogens (Norton, 2000(Norton, ). et al., 1990)).
Therefore, the goal of this study was to determine the incidence of Listeria spp.and L. monocytogenes in In the past 25 years, L. monocytogenes has minced frozen beef, luncheon, and frozen chicken become increasingly important as a food-associated meats as well as in human stools from hospitalized pathogen.control study was conducted.The cases were defined Unlike most other enteric pathogens, L.
as the children, resident in Pediatric Univ.Hospital; monocytogenes is notable for its ability to grow at Assiut Univ., with diarrhea or fever between January refrigeration temperatures.This has considerable and July 2009.A stool culture examined for Listeria spp.significance for food safety, as it means that chilling to The parents of the cases were interviewed, by a single 4º C cannot be relied upon to prevent the growth of the investigator using a standardized questionnaire organism to dangerous levels (Pal et al., 2008).
addressing the family's consumption of, and In addition, because of its ability to survive and purchasing and preparation conditions for, various proliferate at refrigeration temperature, L.
foods such as poultry and beef, and their contacts with monocytogenes may cause disease through frozen people having presented with an episode of diarrhea.foods (Schillinger et al., 1991).Due to its ubiquitous Isolation of Listeria spp.(FAO, 1992): character, L. monocytogenes easily enters the human Enrichment procedures: The initial procedure of food chain and may multiply rapidly (Farber and isolation involves the use of Listeria selective Peterkin, 1991).
enrichment broth (LSEB) to enhance the growth of The standard microbiological methods for Listeria spp.LSEB base consists of trypticase soy identification of Listeria spp.Are laborious and time consuming requiring a minimum of five days to broth with 0.6% yeast extract supplemented with recognize Listeria spp.And about 10 days to identify L. Listeria selective supplement (Hi Media laboratories) monocytogenes by confirmation tests (Amagliani et al., which contains acriflavin-HCL (15 mg/L), nalidixic acid (40 mg/L) and cycloheximide (50 mg/L).Ten grams of 2007) while rapid response should be carried out in samples as well as swabs from human stools were case of confirmation since it is of principal importance to ensure the safety of foods.In the few past years, aseptically added to 90 ml LSEB and mixed thoroughly.progressing in biotechnology has resulted in the All the primary enrichment broths were incubated at development of rapid methods that reduce the analysis 37°C and 30 °C for 24-48 h.time and offer great sensitivity and specificity in the Selective plating: Following the enrichment detection of pathogens.Among these, PCR has been procedure, a loopful of homogenate was streaked onto increasingly used for the rapid, sensitive and specific Listeria selective agar base (Hi Media laboratories)  as vehicle of other pathogenic organisms.The frequent Genomic DNA Extraction : For each Listeria strain, a occurrence of L. monocytogenes in meat and chicken 10-ml culture was grown to mid-log phase in Tryptose may pose a potential risk for consumers (Mahmood et Soya (TSY) broth, and 1 ml of cells was pelleted by al., 2003).Human infections primarily result from centrifugation (13.000 ×g for 5 min).The cell pellets eating contaminated food and may lead to serious and were resuspended in 1ml of sterile water.The potentially life-threatening listeriosis (Posfay-Barbe resuspended cells were re-centrifuged at 12.500 ×g for and Wald, 2004).15 min.The pelleted cells were then used for DNA Of the total of 100 meat and chicken product extraction.
samples examined, 41 (41%) isolates of Listeria spp.Genomic DNA from suspected Listeria strains were recovered.Of these 13 isolates displayed betawas extracted using the Wizard genomic DNA haemolysis on sheep blood agar and positive CAMP purification kit (Promega, USA) as recommended by test.These 13 selected Listeria isolates were further the manufactures.Protocol for Gram positive bacteria, identified using PCR.cellular lyses was carried out by enzymatic fragment The wide application of nucleic acid amplification with lysozyme.DNA samples were stored at -20 °C techniques and the increasing industrial interest until use.
toward rapid methods has led to the development and PCR identification of Listeria monocytogenes : For application of PCR based methods for the detection of L. monocytogenes PCR identification, two primers microbial pathogens in food (Germini et al., 2009).were selected based on the prfA (transcriptional Analyzing the PCR profiles, 5 out of 13 isolated activator of the virulence factor) gene for L.
Listeria strains showed one amplified product (217 bp), monocytogenes as mentioned by (Germini et al., (Fig. 1) that is specific for L. monocytogenes.Thus, L. 2009).Primer sequences used in the PCR are listed in monocytogenes were confirmed in one of minced Table 1.
imported frozen meat examined, two of luncheon All PCR reactions were performed in a final samples and two of frozen chicken legs with the total volume of 25µl using 2µl of extracted DNA as template.
incidence of 5 isolates (5%) from the total 100 Each reaction mixture contained 12.5 µl GoTaq® examined samples (Table 3 and Figure 1).This Green Master Mix (Promega, M7122) 1µl of 500 ?M suggests the presence of a significant public health forward primer (LIS-F);1µl of 500 pM reverse primer hazard linked to the consumption of these meat and (LIS-R) and 8 µl of Ultra-Pure DNase/RNase-Free Distilled Water (Gibco, Grand Island, NY, USA).chicken products sold in Assiut city contaminated with L. monocytogenes.The amplification profile was as follows: pre incubation at 95 °C for 5 min; 40 cycles consisting of The percentage of culture positivity of L. monocytogenes in meat and chicken products in the dsDNA denaturation at 95 °C for 30 s, primer annealing present study is in agreement with the reported at 54°C for 30 s, primer extension at 72 °C for 30 s; final incidences in other countries such as 5.1% in Ethiopia elongation at 72 °C for 5 min.Reactions were thermally (Molla et al., 2004), 4.9% in Belgian meat products cycled in a Techne Cyclogene.
(Uyttendaele, 1999) and 3.6% in processed meat Gel Electrophoresis: All amplification products were Furthermore, detection of L. monocytogenes in When several studies in various countries are foods can be difficult as these bacteria are normally L. monocytogenes found in very low numbers in the presence of a compared, isolation rates seem to heterogenous microflora.The most frequent Listeria vary significantly.This wide variation may be explained in terms of geographic location, isolation methods and isolates from food are L. monocytogenes and L. kinds of media employed (Akpolat et al., 2004).

Occurrence of Listeria species in meat, chicken products and human stools in Assiut city
innocua.Several studies have demonstrated that L. Concerning minced imported frozen meat innocua is found in food more frequently than L. examined in our study, Listeria spp.were isolated from monocytogenes (Walsh et al., 1998).The reasons for 8 (32%) of 25 examined samples.L. monocytogenes the higher frequency of recovery of L. innocua remain occurred in one (4%) and L. innocua in 7 (28%) of unclear yet.However, this may result from either a tested samples, respectively (Tables 2 & 3).naturally higher prevalence or from preferential Similarly, the isolation rate of L. monocytogenes selection of L. innocua during laboratory detection in minced beef samples was 5% in a study in Turkey procedures (Gnanou Besse et al., 2005).conducted by Akpolat et al (2004).Nearly, similar Contamination of the meat with L. monocytoresults obtained by other researchers such as Abd Elgenes generally occurs after the slaughter and may Aziz (2004) (6%) and Marinsek and Grebenc (2002) come from the skin of the animals, the hands of the who isolated L. monocytogenes from 3 of the minced workers, the equipment and the tools used (Marinsek meat samples (6.81 %).
and Grebenc, 2002).On the other hand, higher records were reported Regarding luncheon samples as shown in  Japan and Buncic (1991) detected L. Mohamed andAli, 1999 andSaad et al., 2001).monocytogenes in 69% of minced meat samples in Cross-contamination, which can occur within the Yugoslavia.
environment of food-processing equipment, is It is interesting to note that L. innocua was considered to be a possible source of Listeria isolated predominantly among Listeria spp. in minced contamination in processed meat such as luncheon.L. frozen meat in this study (Table 3).This finding is in monocytogenes is able to attach to and survive on agreement with other studies where L. innocua was the various working contact surfaces (Borucki, 2003).One most common species in raw and cooked meats, while reason may be its ability to form biofilms (Wong, 1998).other Listeria spp.were less frequently (Choi et Occurrence of Listeria species in meat, chicken products and human stools in Assiut city

Table - 3: Incidence of Listeria spp. in meat and chicken products as well as in human stools
Occurrence of Listeria species in meat, chicken products and human stools in Assiut city L.monocytogenes in food in Chile.Intern.J. FoodIt was significantly important for public health toMicrobiol., 70: 175-178.d e t e c t L i s t e r i a s p p. , a n d p a r t i c u l a r l y L .13. De Simon, M.; Tarrago, C. and Ferrer, M. D. (1992): monocytogenes, in meat products sold in Assiut, since Incidence of Listeria monocytogenes in fresh foods in consumers are frequently exposed to these products.Barcelona (Spain).Int.J. Food Microbiol.,16:153-156.Therefore, meat and meat products must be thoroughly 14.Donald, W.W.; Jeffrey, M.F.A. and Ricardo, C. (1991): A cooked or grilled before consumption so L. comparative study of modified versions of monocytogenes is likely to be eliminated.the FDA and USDA methods for the detection of Listeria monocytogenes.J. Food Prot., 54: 669-676.In conclusion, this study has demonstrated the 15.Elgazzar, M.M.M. and Sallam, Kh.I. A. (1997): presence and distribution of L. monocytogenes and Occurrence of Listeria monocytogenes and other other Listeria spp. in a variety of meat and chicken listeria species in meat products.Alexandria Journal of products in Assiut city.The study also suggests the Veterinary Science 13, 4: 415-422.need for improved food safety through the 16.Endang, P.; Radu, S.; Ismail, A.; Kgueen, C.Y. and implementation of hygienic measures at all levels from Maurice, L. (2003): Characterization of Listeria production to consumption.monocytogenes isolated from chicken meat: Evidence of Conjugal transfer of Plasmid-mediated Resistance to of