Filter-based pathogen enrichment technology for detection of multiple viable foodborne pathogens in 1 day

Conventional foodborne pathogen assays currently used in the food industry often require long culture enrichments to increase pathogen levels so they can be detected. Even using sensitive real-time PCR assays, culture enrichment at least overnight is necessary especially for detection of pathogens with slow growth rates such as Listeria monocytogenes. To eliminate this cumbersome enrichment step and detect minute amounts of pathogens within 1 day, filter-based pathogen enrichment technology was developed utilizing a unique combination of glass fiber depth filter and porous filter aid materials to efficiently separate pathogens from food homogenates and avoid filter clogging by food particles. After pathogen immobilization in depth filters, only viable pathogens were selectively collected in a small volume of growth medium via microbial multiplication and migration; nonviable pathogens remained inside the filters. By assaying viable pathogens using real-time PCRs, multiple species of foodborne pathogens were detected, including L. monocytogenes, Salmonella enterica, and Escherichia coli O157:H7, at around 1 CFU/ml or 1 CFU/g in various food samples. This filter-based pathogen enrichment technology is a unique bacterial enrichment alternative to the conventional culture enrichment step and can significantly shorten the time necessary to obtain assay results.     

SIMULTANEOUS DETECTION OF LISTERIA MONOCYTOGENES, STAPHYLOCOCCUS AUREUS, SALMONELLA ENTERICA AND ESCHERICHIA COLI O157:H7 IN FOOD SAMPLES USING MULTIPLEX PCR METHOD

In this study, one multiplex polymerase chain reaction (MPCR) assay was developed for simultaneous detection of four foodborne pathogens, i.e., Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Escherichia coli O157:H7. Five specific primer pairs were designed based on the nucleotide sequences of hemolysin gene (hly) of Listeria monocytogenes, thermostable nuclease gene (nuc) of Staphylococcus aureus, invasion gene (invA) of Salmonella enterica, shiga-like toxin gene (stx) and intimin gene (eae) of Escherichia coli O157:H7 in this assay. The specificity and sensitivity of the MPCR method were validated, and the limit of detection (LOD) of this method was about 10 copies. One cfu/mL each of these foodborne pathogens spiked in practical food samples, i.e., ground meat, beef, pork, fish, shrimp, cheese, canola leaf and cabbage, could be detected simultaneously after 24 h enrichment at a rate of 87.5%, indicating that the established MPCR detection method was effective and suitable for practical use.

PRACTICAL APPLICATIONS

This study presents a quick and effective identification method to simultaneous monitor four foodborne pathogens in food samples. The specificity and sensitivity of this method can be used to unambiguously identify these four foodborne pathogens in practical food samples based on the species-specific genes. Therefore, this detection method is applicable for surveillance measures of these four foodborne pathogens in the food production chain.     

Comparison of a real-time PCR method with a culture method for the detection of Salmonella enterica serotype enteritidis in naturally contaminated environmental samples from integrated poultry houses

Conventional culture methods have traditionally been considered the "gold standard" for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis-specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis-specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.     

A real-time PCR assay for the detection of Salmonella in a wide variety of food and food-animal matricest

Conventional culture methods have traditionally been considered the "gold standards" for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.     

Evaluation of detection methods for screening meat and poultry products for the presence of foodborne pathogens

Rapid and molecular technologies such as enzyme-linked immunosorbent assay (ELISA), PCR, and lateral flow immunoprecipitation can reduce the time and labor involved in screening food products for the presence of pathogens. These technologies were compared with conventional culture methodology for the detection of Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7 inoculated in raw and processed meat and poultry products. Recommended protocols were modified so that the same enrichment broths used in the culture methods were also used in the ELISA, PCR, and lateral flow immunoprecipitation assays. The percent agreement between the rapid technologies and culture methods ranged from 80 to 100% depending on the pathogen detected and the method used. ELISA, PCR, and lateral flow immunoprecipitation all performed well, with no statistical difference, compared with the culture method for the detection of E. coli O157:H7. ELISA performed better for the detection of Salmonella, with sensitivity and specificity rates of 100%. PCR performed better for the detection of Campylobacter jejuni, with 100% agreement to the culture method. PCR was highly sensitive for the detection of all the foodborne pathogens tested except Listeria monocytogenes. Although the lateral flow immunoprecipitation tests were statistically different from the culture methods for Salmonella and Listeria because of false-positive results, the tests did not produce any false negatives, indicating that this method would be suitable for screening meat and poultry products for these pathogens.     

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1-10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1-5 CFU L. monocytogenes per 25g/ml of food sample in 30h. The performance of the assay was compared with the Roche Diagnostics 'LightCycler foodproof Listeria monocytogenes Detection Kit'. Both methods detected L. monocytogenes in all artificially contaminated retail samples (n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.     

单增李斯特菌毒力因子多重荧光PCR法 建立与应用

目的建立同时检测单增李斯特菌(Listeria monocytogenes)及其3种毒力因子的多重荧光PCR快速检测方法,并应用于日常食品的检测。方法根据单增李斯特菌溶血素基因hly A、内化素基因inl A和表面蛋白act A基因的保守序列,分别设计合成特异性引物和探针,优化多重荧光PCR反应体系。对该方法的特异性、敏感性和重复性进行评估。结果该法特异性强、敏感性高,对单增李斯特菌纯培养物的最低检出限410cfu/m L;重复性好,变异系数均小于2%。对84份食品检测结果与传统国标法相符,共检出单增李斯特菌4份,检出率为4.76%。多重荧光PCR检测方法耗时1 h,比传统方法节约2~5 d。4株单增李斯特菌分离株中2株同时含有inl A、act A、hly A 3种毒力基因,另2株为毒力基因act A缺失株,提示目前流行株并非同一来源。结论本研究建立的多重实时荧光PCR方法能同时对单增李斯特菌及其3种毒力因子进行快速检测,且灵敏度高、特异性好,为食源性疾病的病原学检测提供了快速可靠的方法。     

Evaluation of universal preenrichment broth for growth of heat-injured pathogens.

Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis. and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2 degrees C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37 degrees C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (> 10(3) CFU/ ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37 degrees C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation-but not at 6 h-sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.     

Different enrichment procedures for recovery of Listeria monocytogenes from raw chicken samples can affect the results of detection (by chromogenic plating or real-time PCR) and lineage or strain identification

This study aimed to evaluate the effect of different enrichment procedures on the detection of Listeria monocytogenes in food, by a comparison of subculture onto chromogenic agar with real-time PCR. Two different culture media, the primary and secondary enrichment broths of the U.S. Food Safety and Inspection Service (FSIS) method used for PCR detection of L. monocytogenes, were compared for the primary enrichment of retail ground chicken samples. L. monocytogenes was detected after the completion of each enrichment procedure in 63% (complete FSIS procedure) and 60% (plain FSIS secondary enrichment broth incubated for 48 h) of the samples by both culture and PCR, whereas a combination of the results for the two enrichment procedures revealed 86% of the samples to be positive. Most of the samples analyzed contained a mixture of lineage I and II strains, and their ratio varied for each enrichment procedure. This mixture could have a significant effect on the result of detection of L. monocytogenes for each individual sample, explaining the increase in positive samples when the results of the two enrichment procedures were combined. The use of different isolation procedures can affect the specific samples identified as positive and the specific strains isolated.     

Enrichment and DNA extraction protocols for the simultaneous detection of Salmonella and Listeria monocytogenes in raw sausage meat with multiplex real-time PCR

A novel method of DNA extraction and purification was developed and was used in conjunction with a multiplex real-time PCR assay for the simultaneous detection of Salmonella and Listeria monocytogenes in a raw meat sample. The PCR used primers targeting the invA gene of Salmonella and the hlyA gene of L. monocytogenes, and PCR products were detected with a LightCycler on the basis of fluorescence from SYBR Green and melting temperature. The assay allowed the detection of 3 Listeria cells and 4 Salmonella cells per g of the original sausage within 10 h, including an enrichment period of 6 to 8 h.     

Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR

A protocol enabling simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains was devised and evaluated using artificially contaminated fresh produce. Association of Official Analytical Chemists (AOAC)-approved polymerase chain reaction (PCR) detection methods for three human pathogens were modified to enable simultaneous and real-time detection with high throughput capability. The method includes a melting-curve analysis of PCR products, which serves as confirmatory test. The modified protocol successfully detected all three pathogens when fresh produce was washed with artificially contaminated water containing E. coli O157:H7 and S. typhimurium down to the predicted level of 1 to 10 cells/ml and L. monocytogenes at 1000 cells/ml. The ability to monitor several pathogens simultaneously will save time and increase our ability to assure food safety.     

A rapid molecular-based assay for direct quantification of viable bacteria in slaughterhouses

A rapid test for microbial quantification in carcass and environmental swabs that does not require enrichment and provides results in less than 4 h is described here. Steps in the assay include the rapid concentration of bacteria on sponge swabs by vacuum filtration followed by real-time PCR detection. The assay has been applied for the detection of coliforms, Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on carcass swabs and environmental samples in a slaughterhouse-processing line. Comparison of this rapid method with standard culture techniques for coliform counts on beef and pork carcass swabs revealed higher numbers of bacteria (2- to 50-fold) by the rapid test compared with the plate counts. This was due to the detection of all bacteria (live, dead, and non-culturable forms) in the rapid assay. To allow detection of only viable bacteria, concentrated samples were treated with ethidium monoazide (EMA) prior to DNA extraction and real-time PCR detection, thereby preventing the amplification of DNA from bacteria with damaged cell walls and allowing only the DNA from bacteria with intact membranes to be detected. EMA treatment resulted in a significant reduction (P < 0.001) in the number of coliforms detected compared to real-time PCR without EMA treatment. In beef swabs, the counts obtained in EMA real-time PCR were not significantly different (P < 0.08) from the culture counts and the correlation coefficient between the two assays was 0.7385. A lower correlation coefficient (0.402) was obtained with pork swabs. The assay described herein has the potential to be applied on a routine basis to slaughterhouse lines for the detection of indicator organisms or specific pathogens.     

Prevalence of Salmonella enterica, Listeria monocytogenes, and Escherichia coli virulence factors in bulk tank milk and in-line filters from U.S. dairies

The zoonotic bacteria Salmonella enterica, Listeria monocytogenes, and Escherichia coli are known to infect dairy cows while not always causing clinical signs of disease. These pathogens are sometimes found in raw milk, and human disease outbreaks due to these organisms have been associated with the consumption of raw milk or raw milk products. Bulk tank milk (BTM) samples (536) and in-line milk filters (519) collected from dairy farms across the United States during the National Animal Health Monitoring System's Dairy 2007 study were analyzed by real-time PCR for the presence of S. enterica and pathogenic forms of E. coli and by culture techniques for the presence of L. monocytogenes. S. enterica was detected in samples from 28.1% of the dairy operations, primarily in milk filters. Salmonella was isolated from 36 of 75 PCR-positive BTM samples and 105 of 174 PCR-positive filter samples, and the isolates were serotyped. Cerro, Kentucky, Muenster, Anatum, and Newport were the most common serotypes. L. monocytogenes was isolated from 7.1% of the dairy operations, and the 1/2a complex was the most common serotype, followed by 1/2b and 4b (lineage 1). Shiga toxin genes were detected in enrichments from 15.2% of the BTM samples and from 51.0% of the filters by real-time PCR. In most cases, the cycle threshold values for the PCR indicated that toxigenic strains were not a major part of the enrichment populations. These data confirm those from earlier studies showing significant contamination of BTM by zoonotic bacterial pathogens and that the consumption of raw milk and raw milk products presents a health risk.     

多重PCR同时检测食品中4 种细菌与常见霉菌

建立一种同时检测食品中金黄色葡萄球菌、单核细胞增生李斯特氏菌、沙门氏菌、大肠杆菌O157:H7以及霉菌的多重聚合酶链式反应（polymerase chain reaction,PCR）检测方法。根据金黄色葡萄球菌的耐热核酸酶基因（nuc）、沙门氏菌的侵袭正调节蛋白基因（hilA）、大肠杆菌O157:H7鞭毛基因（flic）、单核细胞增生李斯特氏菌的毒力调控蛋白基因（prfA）及霉菌18S?rRNA基因V5区分别设计5?对特异性引物,并对PCR扩增反应体系和扩增条件进行优化,确定获得特异性良好的PCR扩增产物。而后在37?℃对人工污染致病菌的香肠、面包和豆腐进行增菌培养,5?种致病微生物在20?h内的检出限均可达到100?CFU/25?g。本实验建立的多重PCR检测方法适用于食品中4?种细菌与常见霉菌的同时检测,相较于传统检测方法,具有快速、简便、特异性高的优点。     

Novel genomic tools for specific and real-time detection of biothreat and frequently encountered foodborne pathogens

The bacterial genera Escherichia, Salmonella, Shigella, Vibrio, Yersinia, and Francisella include important food safety and biothreat agents. By extensive mining of the whole genome and protein databases of diverse, closely and distantly related bacterial species and strains, we have identified novel genome regions, which we utilized to develop a rapid detection platform for these pathogens. The specific genomic targets we have identified to design the primers in Francisella tularensis subsp. tularensis, F. tularensis subsp. novicida, Shigella dysenteriae, Salmonella enterica serovar Typhimurium, Vibrio cholerae, Yersinia pestis, and Yersinia pseudotuberculosis contained either known genes or putative proteins. Primer sets were designed from the target regions for use in real-time PCR assays to detect specific biothreat pathogens at species or strain levels. The primer sets were first tested by in silico PCR against whole-genome sequences of different species, subspecies, or strains and then by in vitro PCR against genomic DNA preparations from 23 strains representing six biothreat agents (Escherichia coli O157:H7 strain EDL 933, Shigella dysenteriae, S. enterica serovar Typhi, F. tularensis subsp. tularensis, V. cholerae, and Y. pestis) and six foodborne pathogens (Salmonella Typhimurium, Salmonella Saintpaul, Shigella sonnei, F. tularensis subsp. novicida, Vibrio parahaemolyticus, and Y. pseudotuberculosis). Each pathogen was specifically identifiable at the genus and species levels. Sensitivity assays performed with purified DNA showed the lowest detection limit of 128 fg of DNA/μl for F. tularensis subsp. tularensis. A preliminary test to detect Shigella organisms in a milk matrix also enabled the detection of 6 to 60 CFU/ml. These new tools could ultimately be used to develop platforms to simultaneously detect these pathogens.     

单核增生李斯特氏菌近红外快检方法建立与应用

目的建立一种近红外荧光免疫法快速检测单增李斯特氏菌的方法。方法采用近红外荧光染料Dylight800标记单增李斯特菌单克隆抗体,结合免疫侧向流技术制备单增李斯特菌快速检测试纸条。结果整个实验过程仅需45 min,对食品中单增李斯特氏菌最低检测限为500 CFU/mL,与其他5种食源性致病菌均无交叉反应。特异性为89.47%,敏感性为100%。采用近红外荧光免疫层析法和传统方法对30份食品进行检测,结果发现2种方法符合性达93.3%。结论所研制的单增李斯特菌的近红外快检试纸条具有特异性、敏感性较高的特点,适用于食品样本中单增李斯特菌的即时检测。     

Combined PCR and slot blot assay for detection of Salmonella and Listeria monocytogenes

Detection of Salmonella typhimurium and Listeria monocytogenes by the polymerase chain reaction (PCR) assay coupled with slot blot detection was investigated in this study. After being extracted from diluted bacterial culture with the extraction buffer, bacterial DNA was subjected to PCR. The slot blot assay was optimized and used to detect PCR products. The lowest detection level of this method was 10(3) cfu/ml in the original culture media for both pathogens, or 5 bacterial cells in the PCR reaction. Combined with immunomagnetic separation (IMS) to separate and concentrate bacteria from samples, the detection limit could be 40 cfu/ml of bacteria from milk samples. The whole detection procedure was completed within 7 h. After multiplex PCR (amplification of DNA from two different bacteria in the same PCR tube) and slot blot, a detection level of 10(3) cfu/ml was achieved in the simultaneous detection for both pathogens, which was similar to that of individual detection for each pathogen. The combination of PCR and slot-blot seems to be highly sensitive and time-efficient, and is therefore promising for routine use in the detection of Salmonella and L. monocytogenes in food samples such as milk.     

多重PCR检测4种常见食源性致病菌

目的 建立一种多重聚合酶链式反应法(multiplex polymerase chain reaction, MPCR)快速检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌的分析方法。方法 选取金黄色葡萄球菌nuc基因、沙门氏菌SipB基因、志贺氏菌ipaH基因、单增李斯特菌inlA基因作为目标基因, 设计4对PCR引物, 建立并优化多重PCR反应体系, 评价该体系的特异性和灵敏度, 并对人工污染的熟肉样品进行检测。结果 构建的多重PCR方法特异性强、灵敏度高, 人工污染熟肉匀浆中4种致病菌的检出限为103 CFU/mL。结论 构建的多重PCR检测方法能够快速、准确、高效地检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌, 为食源性疾病菌的快速检测提供参考依据。