Rapid polymerase chain reaction/DNA probe membrane-based assay for the detection of Listeria and Listeria monocytogenes in food

We describe the development of polymerase chain reaction (PCR)/DNA probe membrane-based colorimetric assays for the detection and identification of Listeria and L. monocytogenes. PCR primers designed from the 16S to 23S rRNA intergenic spacer region amplified products that were reverse hybridized to membrane-bound oligonucleotide probes specific for Listeria and L. monocytogenes with a detection limit of 1 to 10 CFU/25 ml in inoculated raw and pasteurized milk samples. These qualitative assays have the potential to be integrated into testing laboratories for monitoring the microbiological quality of foods.     

A rapid polymerase chain reaction (PCR)-based assay for the identification of Listeria monocytogenes in food samples.

A rapid and simple assay has been developed which allows specific detection of Listeria monocytogenes within 3.5 h in cultures prepared from suspect food samples and propagated 48 h in selective medium. The assay is based on PCR technology, and uses a specific primer set derived from sequences located down-stream of the hlyA gene. The specificity of the primer set was confirmed by testing 115 L. monocytogenes, 14 L. innocua, 5 L. seeligeri and 4 L. ivanovii isolates. The assay was compared to standard microbiological tests and gave identical results for 83 food samples, including 32 positives. These field trials indicate that the assay developed provides an alternative detection system for L. monocytogenes in foods, which can be used by the food industry.     

Comparative evaluation of culture- and BAX polymerase chain reaction-based detection methods for Listeria spp. and Listeria monocytogenes in environmental and raw fish samples

Two commercial polymerase chain reaction (PCR)-based Listeria detection systems, the BAX for Screening/Listeria monocytogenes and the BAX for Screening/Genus Listeria, and a culture-based detection system, the Biosynth L. monocytogenes Detection System (LMDS), were evaluated for their ability to detect L. monocytogenes and Listeria spp. in raw ingredients and the processing environment. For detection of L. monocytogenes from raw fish, enrichment was performed in Listeria enrichment broth (LEB), followed by plating on both Oxford agar and LMDS L. monocytogenes plating medium (LMPM). Detection of Listeria and L. monocytogenes from environmental samples was performed using LMDS enrichment medium, followed by plating on both Oxford agar and LMPM. A total of 512 environmental samples and 315 raw fish were taken from two smoked fish processing facilities and screened using these molecular and cultural Listeria detection methods. The BAX for Screening/L monocytogenes was used to screen raw fish and was 84.8% sensitive and 100% specific. The BAX for Screening/Genus Listeria was evaluated on environmental samples and had 94.7% sensitivity and 97.4% specificity. In conjunction with enrichment in LEB, LMPM had a sensitivity and specificity for detection of L. monocytogenes from raw fish of 97.8 and 100%, respectively. Use of LMDS enrichment medium followed by plating on LMPM allowed for sensitivity and specificity rates of 94.8 and 100%, respectively, for detection of L. monocytogenes from environmental samples. We conclude that both the BAX systems and the use of LMPM allow for reliable and rapid detection of Listeria spp. and L. monocytogenes. While the BAX systems provide screening results in about 3 days, the use of LMPM allows for L. monocytogenes isolation in 4 to 5 days.     

Evaluation of a polymerase chain reaction-based test for detecting Salmonella spp. in food samples: Probelia Salmonella spp

A commercially available polymerase chain reaction (PCR) kit was evaluated for the detection of Salmonella spp. in food samples. The test combines PCR amplification and sandwich hybridization of the amplified DNA in microtiter plates. The sensitivity and specificity was evaluated with 52 Salmonella strains and 51 non-Salmonella strains and showed that the test was entirely reliable. The threshold sensitivity was 10(2) CFU/ml. The limit of detection of dead cells that determines the minimum detection level of dead cells in food samples was superior to 10(6) CFU/25 g, a level rarely achieved in naturally contaminated samples. After an 18-h pre-enrichment step, the test could detect viable Salmonella in artificially contaminated food samples, even for the lower contamination level (3 CFU/25 g). There was complete agreement between the PCR test and the ISO 6579 bacteriological reference method with artificially contaminated samples. Regarding the accuracy of the results obtained from 253 naturally or noncontaminated foods and from 32 artificially contaminated foods, the agreement percentage was 99.6%. The fidelity of the technique was evaluated in a collaborative study with eight European laboratories and showed a correlation of 98.4%.     

免疫磁分离技术在食源性单增李斯特菌检测中应用的研究进展

食源性单增李斯特菌是李斯特菌属中的唯一能引起人类疾病的病原菌,致死率30%~70%,并严重威胁着人类健康。早期快速准确地检测出食品中可能污染的单增李斯特菌对于减少死亡率非常重要,因此亟需建立一些快速、灵敏和高特异性的检测方法。现有单增李斯特菌的检测方法对未经前增菌的食品样本检测灵敏度较低,限制了这些方法直接用于食品样本中单增李斯特菌的快速检测。免疫磁分离是一种可以短时间内高效富集样本中目的菌的技术,与常用的检测方法结合,可以缩短检测周期,提高检测灵敏度。本文综述了免疫磁分离技术在食源性单核细胞增生性李斯特检测中应用的研究进展。     

免疫磁分离技术在食源性单增李斯特菌检测中应用的研究进展

食源性单增李斯特菌是李斯特菌属中的唯一能引起人类疾病的病原菌,致死率30%~70%,并严重威胁着人类健康。早期快速准确地检测出食品中可能污染的单增李斯特菌对于减少死亡率非常重要,因此亟需建立一些快速、灵敏和高特异性的检测方法。现有单增李斯特菌的检测方法对未经前增菌的食品样本检测灵敏度较低,限制了这些方法直接用于食品样本中单增李斯特菌的快速检测。免疫磁分离是一种可以短时间内高效富集样本中目的菌的技术,与常用的检测方法结合,可以缩短检测周期,提高检测灵敏度。本文综述了免疫磁分离技术在食源性单核细胞增生性李斯特检测中应用的研究进展。      

食品中单核细胞增生李斯特菌株的分离及聚合酶链式反应鉴定

目的 建立单核细胞增生李斯特菌的聚合酶链式反应(PCR)检测方法,了解市售食品中单核细胞增生李斯特菌的污染情况.方法 采集成都市市售生畜肉、生禽肉、熟肉制品、水产品、生食蔬菜以及其他熟食等食品样品共135份,采用李氏增菌肉汤(LB1,LB2)进行初增菌,应用选择性分离培养基PALCAM和在TSA-YE平板上进行分离,利用单增李斯特显色平板进行鉴定;根据李斯特菌的特异性基因iap基因设计引物,采用PCR方法检测所有分离的李斯特菌株;根据单增李斯特菌的特异性基因hly基因和prfA基因设计引物检测单核细胞增生李斯特菌株.结果 135份样品中共检出李斯特菌17株,检出率为12.6％;其中单核细胞增生李斯特菌4株,检出率为3.0％.结论 本研究建立的PCR方法具有特异性,本市市售食品不同程度受到李斯特菌的污染.     

Development and use of polymerase chain reaction for the specific detection of Salmonella Typhimurium in stool and food samples.

Salmonella Typhimurium is one of the most important Salmonella serovars that may cause foodborne disease and human salmonellosis infection. Detection of this organism in the clinical samples of persons with gastroenteritis and the food samples associated with such persons may allow us to trace the cause of disease. Because malic acid dehydrogenase, an enzyme of the citric acid cycle, is common to organisms, the gene (mdh) coding for this enzyme was selected for the design of Salmonella Typhimurium-specific polymerase chain reaction (PCR) primers. By comparison of the mdh gene sequences of Salmonella Typhimurium and other Salmonella serotypes and of some isolates of other genera, two oligonucleotides were designed and used as PCR primers for the specific detection of Salmonella Typhimurium. The molecular weight of the PCR product was 261 bp as expected. Salmonella serovars other than Salmonella Typhimurium and isolates of other genera in the Enterobacteriaceae that is closely related to Salmonella did not generate any false-positive results. When this primer pair was used for the detection of Salmonella Typhimurium cells artificially inoculated into human stool specimens and food samples, such as milk and raw chicken meat, levels as low as 10(0) CFU per 0.1 g of stool specimen or per ml of milk or food homogenate could be detected if an 8- to 12-h preculture step using combined lactose-tetrathionate broth was performed prior to the PCR.     

Rapid detection of Listeria monocytogenes by nanoparticle-based immunomagnetic separation and real-time PCR

The objective of this study was to develop a method combining nanoparticle-based immunomagnetic separation (IMS) with real-time PCR for a rapid and quantitative detection of Listeria monocytogenes. Carboxyl modified magnetic nanoparticles were covalently bound with rabbit anti-L. monocytogenes via the amine groups. Several factors, such as the amount of immunomagnetic nanoparticles (IMNPs), reaction and collection times, and washing step, were optimized, and the nanoparticle-based IMS in combination with real-time PCR was further evaluated for detecting L. monocytogenes from artificially contaminated milk. The cell numbers calculated from the means of threshold cycles (CT) of PCR amplification curves were compared to those from plate counts in order to determine the correspondence degree of quantitative data. The capture efficiency (CE) by plating from IMNP-based IMS was 1.4 to 26 times higher than those of Dynabeads-based IMS depending on the initial cell concentrations inoculated into milk samples. When combined with real-time PCR, L. monocytogenes DNA was detected in milk samples with L. monocytogenes >or=10(2) CFU/0.5 ml. In the range of 10(3) to 10(7)L. monocytogenes CFU/0.5 ml, cell numbers calculated from CT values were 1.5 to 7 times higher than those derived from plate counts. Our results demonstrated that both the use of nanoparticles and the choice of anti-L. monocytogenes in our IMNP-based IMS in combination with real-time PCR has improved the sensitivity of L. monocytogenes detection from both nutrient broth and milk samples.     

Multiplex PCR for the simultaneous detection of Salmonella spp. and Salmonella Enteritidis in food

Multiplex PCR assay (mPCR) for the detection of Salmonella spp. and S. Enteritidis was developed in this study using artificially contaminated chicken carcasses. The assay showed 100% specificity to detect approximately 1 CFU of Salmonella in 10 g of chicken skin after non‐selective enrichment. The mPCR was evaluated in Minas cheese, fresh pork sausage and chicken carcasses commercially available. Salmonella spp. was detected in nine of sixty‐six chicken carcasses, five of fifty‐two cheese samples, and five of fifty‐two sausage samples. The serovar Enteritidis was detected in two samples of contaminated sausage. The mPCR results were confirmed by conventional culture and biochemical identification of the isolates. Serotyping confirmed the presence of S. Enteritidis in sausage samples and showed contamination by serovars Schwarzengrund and Montevideo in chicken carcasses.     

Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 10(3) CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.     

Development of a multiplex real-time PCR method for simultaneous detection of Salmonella enterica, Shigella flexneri and Listeria monocytogenes in processed food samples

The present work is focused on the development of a TaqMan multiplex real-time PCR method for the detection of Salmonella, Shigella and L. monocytogenes in seafood, meat and ready-to-eat products. The aim of this study is to detect the three pathogens in one single test including an enrichment medium for the simultaneous growth of the bacteria of interest and an Internal Amplification Control (IAC) to monitor PCR inhibitors. For this purpose, three genes were selected, invA for Salmonella, ipaH for Shigella and hlyA for L. monocytogenes. Also, no. 17 broth without dextrose and further modified by adding Tween 80 was used for the enrichment step. Specificity of the method was checked against a panel of 24 non-target bacterial strains. RT-PCR efficiency obtained for the simultaneous amplification of all three pathogens was 102.5% for Salmonella, 108.9% for Shigella and 106.4% for L. monocytogenes. The limit of detection (LOD) was evaluated in seafood, meat and ready-to-eat products, being established within 3 and 22 cfu in 25 g of sample for the three bacteria analyzed. Seventy-eight samples were analyzed with multiplex RT-PCR including spiked and natural samples collected from different laboratories. Even though several RT-PCR methods have been developed for the detection of Salmonella, Shigella and L. monocytogenes, as far as we know this is the first method developed for the simultaneous detection of these three pathogens, coupling RT-PCR with an enrichment in the same broth and being tested in a wide range of different processed food samples with a low LOD. The application of this method can significantly reduce costs and time of analysis in laboratories, what would be reflected in a faster response in those risk situations when they are detected.     

Evaluation of antibodies for immunomagnetic separation combined with flow cytometry detection of Listeria monocytogenes

Four polyclonal anti-Listeria antibodies were evaluated for the detection of Listeria monocytogenes in direct and indirect assays using immunomagnetic separation with flow cytometry. The efficiency of immunocapturing using magnetic beads was also determined. None of the tested antibodies exhibited sufficient specificity or avidity to allow sufficient separation and detection of L. monocytogenes for a useful test that differentiated between negative (without cell) and positive (with cell) samples. Plating results confirmed that cells were captured with Dynabeads anti-Listeria and magnetic beads coated with goat anti-Listeria antibody with recovery ranging from 7 to 23%. Fluorescent-labeled polyclonal antibodies used in this study were not sufficiently specific to allow the detection of L. monocytogenes cells captured by the beads.     

Application of a multiplex polymerase chain reaction assay for the simultaneous confirmation of Listeria monocytogenes and other Listeria species in turkey sample surveillance

A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.     

食品中单核细胞增生单核细胞增生李斯特菌的重组酶聚合酶扩增检测方法的建立

目的 建立食品中单核细胞增生李斯特菌的重组酶聚合酶扩增(RPA)检测体系。方法 针对单核细胞增生李斯特菌的特异性基因hlyA,筛选出一组特异性强且高效的引物,建立单核细胞增生李斯特菌的RPA检测体系,并进行特异性、灵敏度检测。结果 建立的RPA方法在37 ℃恒温反应仅需30 min,能够特异地检测单核细胞增生李斯特菌,最低模板浓度可低至0.5 ng/μL。人工染菌试验显示,该RPA体系可以有效扩增出每2.5 g样品中人工染菌10⁴ CFU样品中的单核细胞增生李斯特菌。结论 RPA等温扩增方法特异较强、灵敏度较高,具有操作简便、不需要昂贵仪器、常温进行扩增反应等优点,适用于现场检测以及在基层实验室推广应用。     

Detection of Salmonella spp. in tropical seafood by polymerase chain reaction

The incidence of Salmonella spp. in tropical seafood was studied using standard microbiological techniques and polymerase chain reaction (PCR). Six of 20 finfish (30%), 4 of 20 clams (20%) and 1 of 20 shrimp (5%) were positive by culture techniques and by PCR. In a comparative study of different selective enrichment broths and selective plating media, more than one enrichment broth and selective agar were found to be necessary for efficient detection of Salmonella from seafood. Selenite cystine broth (SCB) was found to be more efficient compared to tetrathionate broth (TTB) while both bismuth sulfite agar (BSA) and hektoen enteric agar (HEA) were equally effective as selective plating media for fish. In the case of clams, HEA was found to be more effective. The presence of Salmonella spp. could be detected by PCR amplification of DNA extracted directly from the enrichment broths. In two cases, enrichment broths that were positive by PCR did not yield Salmonella by conventional methods.     

Detection of Listeria monocytogenes in salmon using the Probelia polymerase chain reaction system

A validation was conducted on the performance of a commercially available polymerase chain reaction (PCR) kit (Probelia) in comparison with International Organization for Standardization (ISO) method 11290-1 (adopted as an Australian New Zealand Standard Method, AS/NZS 1766.2.16.1:1998) for the detection of Listeria monocytogenes in salmon samples. The validation was conducted following the guidelines of an Australian New Zealand Standard (Guide to Determining the Equivalence of Food Microbiology Test Methods, Part 1, Qualitative Tests, AS/NZS 4659.1:1999), which adopts an approach similar to that recommended by the Association of Analytical Communities Microbiology Method Validation Program for Performance Tested and Peer Verified Methods. The validation study involved the use of five cultures of L. monocytogenes, each challenged at a single level of inoculation into five different types of salmon samples. A total of 60 salmon samples (30 unchallenged and 30 challenged) were tested using both the PCR method and the ISO method. Results from this study indicated that the Probelia PCR method is equivalent to the ISO method. In addition, the detection sensitivity of the Probelia PCR system was determined as approximately 0.5 CFU per PCR assay (equivalent to 20 CFU/ml broth culture) for a pure culture of L. monocytogenes. The Probelia PCR method offers the advantage of detecting L. monocytogenes to genetic specificity within 48 to 50 h, whereas the ISO method requires 5 days for negative results with additional days for confirmed positive results by the use of other biochemical and cultural tests.     

Rapid detection of Listeria monocytogenes using fluorescence immunochromatographic assay combined with immunomagnetic separation technique

To ensure the safety and quality of food ingredients, especially meat and dairy products, a high‐throughput, rapid and sensitive method to detect Listeria monocytogenes (LM) is always on a high demand. In this work, a specific induction method to enrich and detect LM based on fluorescence immunochromatographic assay (FICA) combined with immunomagnetic separation (IMS) techniques has been developed. The immunomagnetic‐beads (IM) beads were obtained through functionalised magnetic microspheres and the conjugated reaction between the carboxyl on beads surface and amino groups of antibody. The prepared IM‐beads could be used for rapidly enriching pathogenic bacteria with fewer steps, resulting in a high specificity for four pathogenic serotypes and about a 40‐fold improvement of detection limit compared with FICA only. In addition, this method was successfully applied in LM detection in sausage, pork and milk samples with a potential for further application in rapid on‐site detection of pathogenic bacteria.     

Rapid detection of stressed Salmonella spp. in dairy and egg products using immunomagnetic separation and PCR

The rapid detection of an average of 5.9 stressed Salmonella cells in 25 g of food product using immunomagnetic separation (IMS) and PCR is described. For pasteurised egg yolk, egg yolk powder, ice-cream, whole egg, egg white and cheeses made from pasteurised milk PCR was applied after 16 h of preenrichment in buffered peptone water (BPW) using IMS and alkaline lysis as sample preparation method. For whole egg and egg white the BPW was supplemented with iron. For milk powder, and raw milk cheeses, the 16-h preenrichment in BPW was followed by IMS and a 4-h enrichment in Rappaport-Vassiliadis broth. In the latter case, PCR was applied on the enrichment medium after centrifugation and alkaline lysis. For PCR the primers ST11 and ST15 (Aabo et al., 1993) producing a fragment of 429 bp were used. An internal PCR control, designed to be co-amplified with the target DNA using the same primers but producing a smaller fragment of 240 bp, was used.