RAPID DETECTION OF LISTERIA MONOCYTOGENES IN GROUND TURKEY BY IMMUNOMAGNETIC SEPARATION AND POLYMERASE CHAIN REACTION

The aim of this study was to determine the prevalence of Listeria monocytogenes in packaged fresh ground turkey in Turkey using immunomagnetic separation (IMS) as a selective enrichment step in method and polymerase chain reaction (PCR). A total of 180 ground turkey samples were collected during a 1-year period. Thirty-two (17.7%) of the samples contained L. monocytogenes, 24 (13.3%) contained Listeria innocua, 7 (3.8%) had Listeria ivanovii and 5 (2.7%) had Listeria seeligeri by means of IMS-based cultivation method. A PCR assay was performed, based on hlyA gene-specific primers. In all L. monocytogenes isolates, hlyA gene was confirmed, indicating that the correlation between IMS-based cultivation and PCR methods was 100%. The results suggest that the prevalence of L. monocytogenes in ground turkey is relatively high in Turkey and that ground turkey should be produced under appropriate hygienic and technological conditions for the prevention of public health hazards.

PRACTICAL APPLICATIONS

Using fast and reliable methods to detect and identify foodborne pathogenic bacteria, including Listeria monocytogenes , is important to detect the risk of contaminated product and protect public health. In some ways it is time-consuming to isolate and identify the pathogenic microorganisms from food products using conventional techniques. Different methods or techniques can be used both for redounding the isolation chance and to gain time for this purpose. Immunomagnetic separation (IMS) and polymerase chain reaction (PCR) techniques are effective and rapid methods for separation, detection and confirmation of Listeria spp. from foods. In this study rapid, specific and sensitive IMS method was used to determine the prevalence of L. monocytogenes in fresh ground turkey and PCR technique was used for the verification of the L. monocytogenes isolates.     

快速检测猪肉中单核细胞增多症李氏菌PCR试剂盒的研制

快速检测猪肉中单核细胞增多症李氏菌的ＰＣＲ试剂盒与分离培养法平行检测１５６份猪肉样品,结果ＰＣＲ的阳性率为５．８％,分离培养法的阳性率为３．８％,两者符合率为９７％。实验证明,该试剂盒具有良好的特异性和可重复性,可测出样品中至少３２ＯＣＦＵ细菌,并在２ｄ内取得结果,具有推广应用价值。     

AFFINITY CONCENTRATION OF LISTERIA MONOCYTOGENES CELLS ON CONCANAVALIN A-COATED POLYESTER CLOTH AND SUBSEQUENT DETECTION BY THE POLYMERASE CHAIN REACTION

The Canavalia ensiformis lectin concanavalin A (con A) was immobilized on macroporous polyester cloth to form an inexpensive high surface area adsorbent (con A-cloth) for the affinity concentration of bacterial cells in aqueous suspensions. When the con A-cloth was packed into a 1 ml pipette tip, the resulting mini-column could be used to concentrate large volumes of dilute L. monocytogenes cell suspensions, followed by the polymerase chain reaction (PCR) amplification of L. monocytogenes-specific hly A sequences from lysates of the captured cells. This improved the effective sensitivity of the PCR as compared to the assay of L. monocytogenes in unconcentrated suspensions. Several enrichment broths (Fraser Broth, Listeria Enrichment Broth and Brain Heart Infusion Broth) were found to be inhibitory to the PCR of L. monocytogenes when introduced directly into the reaction mixture. This inhibitory effect was completely eliminated when the L. monocytogenes cells were captured on the con A-cloth and washed to remove the enrichment broth components prior to performing the PCR. Since the lectin con A is reactive with a broad variety of Gram positive and Gram negative bacteria, this simple and inexpensive affinity concentration method should be applicable to the PCR detection of other pathogens in enrichment cultures.     

RAPID DETECTION OF LISTERIA MONOCYTOGENES USING A REFLECTANCE COLORBMETRIC METHOD WITH MEMBRANE FRACTIONS FROM OXIDATIVE BACTERIA

Membrane fractions obtained from Escherichia coli, Gluconobacter oxydans, and Acetobacter xylinum significantly stimulated rapid growth of Listeria monocytogenes (strains 101, 103, and Scott A) under atmospheric conditions of the OmniSpec Bioactivity System. L. monocytogenes demonstrated a shorter lag phase and faster growth compared to culture systems without the membrane fractions as determined by shorter color detection times, the durations of time for a significant color change to occur. The growth stimulating effect increased as the concentrations of membrane fractions increased. The use of membrane fractions to recover facultative Listeria spp. also lowered the detection limit of the method to < 10 cells/ml by increasing small cell numbers to the detectable level (10⁷ cells/m) faster under aerobic conditions. The application of membrane fractions with the OmniSpec Bioactivity System shortened the detection time for the bacteria by 0.5–10 h, depending on the specific membrane and initial cell concentrations, compared to the conventional OmniSpec method. This method is very useful as a tool for studying food microbiology. Membrane fractions produced in our laboratory effectively dissipated oxygen in Fraser broth and had growth-enhancing activities comparable to that of commercial Oxyrase^TM.     

COMPARISON OF THREE POLYMERASE CHAIN REACTION-BASED METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN FOOD

Three PCR-based methods for the detection of Listeria monocytogenes in food (BAX for Screening, Probelia and a method according to Kaclíková et al. (2003) were compared on the basis of the determination of detection limits for 15 artificially contaminated food products. Detection limits of all methods for all samples were 10⁰ cfu per 10 g, with the exception of three cheese samples which did not produce valid results because of the inhibition of Probelia PCR. Detection limits for nonviable L. monocytogenes cells were sufficiently high ( 10⁹ cfu per 10 g) for BAX and the method according to Kaclíková et al. (2003), but considerably low ( 10⁶ cfu per 10 g) for Probelia. The results demonstrate that BAX for Screening as well as the Kaclíková et al. (2001) method fulfill the sensitivity requirements for a rapid alternative method for the detection of L. monocytogenes in food, which would be equivalent to the standard method EN ISO 11290–1.     

DETECTION OF LISTERIA IN FOOD AND ENVIRONMENTAL SAMPLES BY IMMUNOMAGNETIC BEAD CAPTURE AND BY CULTURAL METHODS

Detection of Listeria by capture on antibody-coated magnetic beads has been shown to decrease test time and improve sensitivity, relative to cultural methods, in a study of spiked environmental samples (Mitchell et al. 1993). In this study, immunomagnetic capture was compared to standard cultural methods for detection of Listeria in a broad range of spiked and naturally contaminated food and environmental samples. Immunomagnetic capture was at least as sensitive as cultural methods for detection of Listeria in seafood, meats, dairy foods, and environmental samples. It was possible to determine the number of Listeria present in a sample, because immunomagnetic capture was carried out directly from the sample, without enrichment. These quantitative results were produced within 24 h, while cultural methods required 6–14 days to produce a qualitative result. Immunomagnetic capture was thus more rapid and as sensitive as standard cultural methods for detection of Listeria in the food and environmental samples tested.     

SUITABILITY OF SELECTIVE MEDIA FOR RECOVERY AND ENUMERATION OF SUBLETHALLY HEAT- AND ACID-INJURED LISTERIA MONOCYTOGENES

The suitability of PALCAM and modified Oxford (MOX) agars for recovering sublethally heat- and lactic acid-injured Listeria monocytogenes was investigated. L. monocytogenes LM101M, LM103M (meat isolates), and Scott A were suspended in tryptose phosphate broth (TPB), heated for up to 40 min at 54C, and surface plated onto tryptose phosphate agar (TPA), TPA + 4% NaCl (TPAS), PALCAM, and MOX. TPA and TPAS were used to determine total viable and sublethally injured populations, respectively. Heat-injured LM103M was recovered in the highest numbers on all media, followed by Scott A and LM101M (P<0.01). TPA allowed best recovery of all test strains, followed by PALCAMand MOX which were not different, and TPAS (P<0.01). For acid-injury studies, uninjured and heat-injured (54C for 20 min) test strains were suspended in phosphate-buffered TPB + 0.85% lactic acid (bTPBLA) at 25C for up to 24 h and plated as described above. Uninjured and heat-injured L. monocytogenes were recovered better from bTPBLA on MOX than on PALCAM (P<0.05). Heat injured L. monocytogenes LM103M was recovered better than LM101M but similar to Scott A on MOX and PALCAM (P<0.05), whereas Scott A was recovered similarly to LM101M and LM103M on MOX and PALCAM (P>0.05). Acid-injury of L. monocytogenes LM103M was enhanced by prior heat stress.     

A COLLABORATIVE STUDY FOR DIRECT ENUMERATION OF LOW LEVELS OF LISTERIA MONOCYTOGENES FROM VARIOUS FOODS

A direct plating method for the enumeration of low levels of foodborne Listeria monocytogenes was evaluated in a collaborative study involving 18 laboratories across Canada. Shrimp, coleslaw, ice cream and wieners were inoculated with low levels (5 × 10² and 10³/g) of L. monocytogenes and shipped to participants. Foods were diluted and then plated onto either lithium chloride phenylethyl and moxa-lactam agar (LPM), Oxford agar (OXA), modified Oxford agar (MOX) or Palcam agar (PAL). Recovery was good for all foods, except coleslaw. Of the four plating media tested, all were more or less equivalent in their ability to recover colonies for enumeration, except that more colonies were enumerated on LPM than on PAL agar. Recovery of L. monocytogenes ranged from <50 to 1250 cfu/g for wieners, <50 to 800 cfu/g for shrimp, <100 to 1440 cfu/g for ice cream and <50 to 700 cfu/g for coleslaw. Results indicate that the direct plating method can be used for the recovery of low levels of Listeria monocytogenes in Category 3 foods, as presently suggested for use in the Canadian Listeria compliance guide.