Listeria spp. are broadly distributed in the environment. They can be isolated from soil and water Jersik, et al. [1]. A wide range of animal species can become contaminated by L. monocytogenes, including mammals, domesticated animals, pets, fish, birds, and crustaceans Alsheikh et al. [2].

In mammals, L. monocytogenes can cause premature births and meningoencephalitis Aznar et al. [3]. L. monocytogenes is a concern to public health because of its capability to survive or even grow under harsh conditions [4,5]. The increasing rate of L. monocytogenes involvement in food-born outbreaks has led to the requirement for a rapid method for testing food products. Almost all cases of listeriosis tend to be food-born, and a number of food items can become contaminated by L. monocytogenes, including raw chicken meat, raw minced meat, soft cheese, and fish. Most of these items are widely consumed in Iraq’s Kurdistan and Duhok provinces.

A number of molecular biological methods have been described for detection of L. monocytogenes, including DNA probes and PCR techniques. Direct detection of L. monocytogenes in food products by PCR has been reported in several cases Bansal et al. [6], Manzano et al. [7], Agersborg et al. [8], O’Connor et al. [9] and Hudson et al. [10].

To our knowledge, there is no published data regarding L. monocytogenes prevalence in food samples from Duhok province. In fact, there are no official data of food-born listeriosis recorded in Duhok, as L. monocytogenes is rarely tested for in food samples. The objective of the present research was to assess the use of PCR for the detection of L. monocytogenes in food products, and to determine the prevalence of Listeria spp. in food products in Duhok, by using molecular and biochemical methods.

Samples (n=150) of different food products, including chicken (n=50), minced meat (n=50), and cheese (n=50), obtained from different supermarkets, restaurants and veterinary quarantine centres in Duhok province, were tested. All samples had been correctly stored, and were placed in separate sterile plastic bags to prevent spilling and cross contamination. The samples were brought to the laboratory on crushed ice, and were kept at 4°C until testing (within 4 h).

Food samples were analysed for the presence of Listeria spp. using a selective enrichment and isolation protocol, as recommended by the United States Department of Agriculture (USDA) [11].

Microbiological investigation

Samples (25 g) were added to 225 mL of ½ strength Fraser broth (Conda, Spain), to obtain a 1:10 dilution. All samples were homogenized (30-60 sec) and incubated (30°C, 24 h). From this primary enrichment, 0.1 mL was then inoculated into 10 mL of Fraser broth, and incubated (37°C, 48 h) with shaking. A loopful of the subsequent culture was streaked on the surface of different agars (PALCAM listeria agar (Conda, Spain) with supplement, and OXFORD agar (Lab UK) with X122 supplement). Agar plates were then incubated (37°C, 48 h) and observed (at 24 to 48 h incubation) for suspected colonies (greyish colonies surrounded by black halos, with sunken centres with possible greenish sheen on Oxford, or black colonies on PALCAM). The following tests were used for confirmation; Gram's stain, motility test, catalase and oxidase reactions, and qPCR.

DNA Extraction

Listeria DNA was extracted from a sweep of several colonies grown on PALCAM, using the boiling method. One loop of Listeria from the agar plates was suspended in 100 μL of sterile de-ionized water in a 1.5 mL microcentrifuge tube and vortexed, boiled (95-100°C, 10 min), and centrifuged (10,000 × g, 10 min). The supernatant was used as a DNA template for PCR Nessa et al. [12]. Purification of DNA was achieved by using a genomic DNA purification kit (Qiagen, Germany), according to the manufacturer’s instructions. The DNA was measured with the Nanodrop Spectrophotometer QIAxpert (QIAGEN), and stored at 4°C.

Real-Time PCR

Screening of suspected Listeria was conducted with a Food proof^® Listeria detection kit (Biotecon Diagnostics, Catalog), which uses real-time PCR for the qualitative detection of Listeria spp. This analysis was performed with a final volume of 25 μL of reaction mixture in each well of a 96-well plate, containing 20 μL of PCR Master Mix (provided) and 5 μL of DNA template, with positive and negative controls also being run. DNA was then amplified in the standard mode running (2 h) on a 7500 fast real-time PCR machine (Applied Biosystems, Foster City, CA, USA). Amplification conditions were optimized Table 1. The amplification results were analysed during the last 50 cycles of the amplifications, using the 7500 fast software V.1.4.0 (Applied Biosystems).

Table 1: RT-PCR conditions and cycling for L. monocytogenes detection.

Of the 150 tested samples, 13.3% were presumptively positive Listeria spp. by the culturing method, while 8% were confirmed as L. monocytogenes by PCR. All samples were initially streaked on Palcam and Oxford agar. On Palcam, which is a selective media recommended for the isolation of L. monocytogenes from foods, Listeria colonies appeared grey-green with black–sunken centers and a black halo Figure 1. Twenty putative L. monocytogenes colonies were confirmed as Listeria spp. by being positive for catalase and motility, and negative for oxidase Table 2. Listeria was found in 3 samples of cheese (6%), 6 of chicken (12%), and 11 of meat (22%).

Table 2: Microbiological and Biochemical tests of L. monocytogenes.

Figure 1: L. monocytogenes colonies on Palcam agar (arrows) appears as grey-green with black–sunken centres and a black halo colonies.

Confirmation of L. monocytogenes by real-time PCR

Real-time PCR analyses of the 20 suspected L. monocytogenes samples are shown in Figure 2. The majority (12) of samples were confirmed as positive Table 3.

Table 3: Number of Positive sample according to real-time PCR.

Figure 2: Amplification plot of L. monocytogenes fluorescence versus cycle number. The figure shows that the suspected L. monocytogenes sample amplifying. No amplification in negative and positive controls, but internal control is worked.

Listeria monocytogenes is a highly pathogenic bacterium, contaminating a wide range of food products, and with a high mortality rate. From a total of 150 samples of various types of food products from Duhok province, Iraq, soft cheese 1 (2%), raw chicken 4 (8%), and red minced meat (Lamb) 7 (14%), were positive for L. monocytogenes by PCR. These results indicate that meat products are considerably more likely to be contaminated with L. monocytogenes than other food products. This could be due to preparing and processing minced meat (used, for example, in kebabs) in poor hygienic conditions Akya et al. [13]. Although other factors such as equipment and food additives can also have a role in contamination, kebabs tend to be well cooked prior to consumption, reducing the chance of acquiring listeriosis in this kind of food Akya et al. [13].

Our results showed differences when compared to other studies. For example, a research was conducted by [14] in Isfahan, Iran on various food products including dairy products, meat, and ready-to-eat food found a 4.7% contamination rate with Listeria spp, which is lower than our result (16%) [14]. Interestingly [15] also found that the highest contamination rate was in meat products.

Regarding chicken meat, our results showed the lowest incidence (4%) of samples containing Listeria spp. These values are in agreement with a study performed by Mahmood and his colleagues Mahmood et al. [16]. However, the occurrence of L. monocytogenes in the current research was lower than that found in work carried out by Goh, et al. [17]. Another research conducted by Ayaz et al. [18], showed a higher incidence of L. monocytogenes in chicken meat (20.4%), although the numbers of samples were higher compared to those for the current study Ayaz et al. [18].

In soft cheese samples, we show that the prevalence of Listeria is not significant (2%). Our results were contradicted in another study conducted in Jordan by Osaili et al. [19] who reported approximately 30% L. monocytogenes sample contamination rate. It should be noted that the contamination rate of Listeria in dairy products in our study was consistent with the results of [14]. A low level (1.1%) of L. monocytogenes contamination was found in soft cheese by [13]. Those results are different from the ones obtained in our study. This might be due to contamination during the process of cheese-making, as well as the effectiveness of pasteurization. The acceptable contamination rate of L. monocytogenes ranges between 10% and 15.3% for cheese and raw milk [20,21]. A research was done by Turgay et al. [22], mainly focused on detection of E. coli O157 strains in cheese samples in Duhok area, found that the prevalence of the E. coli O157 was 4%. This shows that the E. coli O157 rate in cheese is double than that compared to L. monocytogenes in the same food source.

Although our results indicate that L. monocytogenes is present in food products in Duhok governorate, the contamination level of this pathogen was comparatively low compared to other foodborne pathogen. While it was clear that meat products showed the highest level of contamination (14%), cheese was least contaminated (2%), with chicken (8%) being intermediate between these. PCR is often considered more dependable than conventional identification methods, since it is based on stable genotypic characteristics rather than relying on biochemical or physiological traits. According to this study, hygienic conditions are highly recommended in order to minimise the presence of L. monocytogenes in foods. We recommend that further research on L. monocytogenes in Duhok is carried out, in order to provide a better background of contamination rate and the routes of transmission for this bacterium.