Enzyme-linked immunoassays for the detection of Salmonella spp.: a comparison with other methods

The first enzyme immunoassay for Salmonella was reported in 1977 and since that time several enzyme-linked immuno assays (ELISAs) have been developed, using either polyclonal or monoclonal antibodies that will detect most Salmonella serotypes. Two of these kits have been declared official first status by the Association of Official Analytical Chemists (AOAC). In comparison with a culture method used in collaborative studies, the total assay time is reduced by 2 days and statistical analysis of the data indicated no significant difference. The main problem related to all methods other than traditional culture methods is the occurrence of false-positive and/or false-negative results. False-positive ELISA results can be eliminated by using (combinations of) highly specific monoclonal antibodies. Good enrichment procedures are very important to be sure that the detection limit of approx. 10(5) cells/ml will be reached. In the future even better limits of detection may be achieved by using enzyme amplification or chemiluminescence to decrease the number of false-negative results.     

Occurrence of Salmonella spp. in samples from pigs slaughtered for consumption: A comparison between ISO 6579:2002 and 23S rRNA Fluorescent In Situ Hybridization method

Contamination of pork products during slaughter represents an important vehicle for Salmonella spp. dissemination to humans. Salmonellosis poses an important risk for public health and presents an important economic issue to pork producers. This study aimed to evaluate the occurrence of this foodborne pathogen in pork carcasses and risk tissues (ileum, ileocolic and mandibular lymph nodes and tonsils) by two methods: the reference culture method (ISO 6579:2002) and a rapid Fluorescent In Situ Hybridization (FISH) method.The culture method identified the presence of Salmonella spp. in 13.7% of the samples, while the FISH technique revealed that 38.2% of the samples were positive. From these FISH positive samples, only 58 were concordant to the positive results obtained by the culture method.These results confirm the potential risk that pork represents in salmonellosis transmission, suggesting that additional measures should be taken during evisceration practices and extraction of tonsils and mandibular lymph nodes after slaughter, in order to achieve a better control of Salmonella contamination during slaughter. The FISH method showed to be a rapid screening tool for Salmonella spp. detection in pork samples.     

Sequencing of an internal transcribed spacer region of 16S-23S rRNA gene and designing of PCR primers for the detection of Salmonella spp. in food

DNA sequences of an internal transcribed spacer (ITS) region for 40 Salmonella serovars were determined and compared with ITS sequences of Salmonella spp., and non-Salmonella spp. already available on the GenBank database. From such comparison, two Salmonella-specific ITS based PCR primers, ITSF and ITSR, were designed. When Salmonella strains with various serotypes were PCR assayed with primers ITSF/ITSR, all generated PCR products with molecular weight bands equal to 312 bp. On the other hand, 48 non-Salmonella isolates, including strains of Enterobacteriaceae and other food pathogens generated negative results. Detection limits of this PCR method was 1-9 CFU per assay. These PCR primers were used for the detection of Salmonella cells in artificially contaminated foods, including chicken meat and whole milk. The detection limit was 1-9 x 10(3) CFU per assay. With an 8-h enrichment step performed prior to the PCR assay, however, the detection limit became 1-9 CFU per gram of the food sample.     

Identification of Bacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. with 16S ribosomal DNA-based oligonucleotide array hybridization

Rapid identification of the genus and species of bacteria in foods and clinical specimens is important. In this report, DNA sequences of bacterial 16S rDNA were used to develop the oligonucleotide array for the identification of bacterial strains of Bacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. Most of these bacterial strains may cause food-borne outbreaks or sporadic cases. A rapid (<4 h) detection method that used universal PCR primers to amplify the variable regions of bacterial 16S rDNA, followed by reverse hybridization of the PCR products, which were biotin labeled, to the oligonucleotides arrayed on the chip was developed. Fifteen oligonucleotide probes were selected and spotted on the nylon strip to determine the array hybridization patterns. It was successful in discriminating Bacillus spp., E. coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. with identification, in general, to the genus level, not species level. As 182 randomly selected strains were assayed, the detection rate was found higher than 98%. Except for 3 strains, the remaining 179 strains were correctly identified and no cross reactions were observed. These 179 strains generated five hybridization patterns. Adding more oligonucleotide probes to the array may allow the detection of more bacterial genera and species without significantly increasing the complexity or cost.     

Salmonella spp. shedding by alberta beef cattle and the detection of Salmonella spp. in ground beef

Breeder cows, cattle recently arrived at feedlots, and cattle about to be shipped for slaughter were tested for Salmonella spp. No Salmonella spp. were detected in fecal samples from breeding cows. Nineteen of 1,000 (1.9%) fecal samples from recently arrived feedlot cattle were positive for Salmonella spp. compared to only 2 of 1,000 (0.2%) fecal samples taken within 2 weeks of slaughter. The positive fecal samples were collected in 5 of 50 (10%) "recent arrival" pens tested and in 1 of 50 (2%) pens tested within 2 weeks of slaughter. The serotypes isolated were Salmonella Agona, Salmonella Enteritidis, Salmonella Typhimurium DT104, and Salmonella 4,5,12:i:-. Ground beef samples purchased from retail outlets throughout Alberta were processed for Salmonella spp. Thirteen of 1,002 (1.3%) samples were positive for Salmonella spp. The serotypes isolated from ground beef were Salmonella Anatum, Salmonella Heidelberg, Salmonella Montevideo, Salmonella Typhimurium, Salmonella Typhimurium var. Copenhagen, and Salmonella Rough-O:i:1,2. The antibiotic resistance and pulsed-field electrophoresis gel macrorestriction patterns of all isolates were compared.     

A comparison of sample weight and culture methods for the detection of Salmonella in pig feces

Five protocols were compared to determine the combined effects of different sample weights and culture methods for the recovery of Salmonella from 310 pig cecal samples taken in abattoirs as part of the Canadian Integrated Program for Anti-microbial Resistance Surveillance. Sample weights evaluated were 1 and 10 g. Culture methods used with each sample weight were modified semisolid Rappaport-Vassiliadis agar (MSRV) and brilliant green agar with sulfa and novobiocin (BGSN) and xylose-lysine-tergitol-4 agar (XLT4). A preliminary sample preparation step in saline was also evaluated using a 10-g sample and MSRV. The Salmonella recovery rate varied from 20% for the saline MSRV 10-g protocol to 32% for the MSRV 10-g and the BGSN-XLT4 10-g protocols. A good agreement (K > 0.8) was observed between pairs of protocols except whenever the saline MSRV 10-g and the MSRV 1-g protocols were compared. Larger samples (10 g) yielded higher detection of Salmonella than 1-g samples for the MSRV protocol (32 versus 25%), whereas the differences were not statistically significant for the BGSN-XLT4 protocols. Protocols using the BGSN-XLT4 agar yielded higher detection rates of Salmonella compared with MSRV with 1-g samples (30 versus 25%), whereas it was equivalent with 10-g samples. Considering a greater recovery rate, the ease of use, and a better time and resource efficiency, the MSRV 10-g protocol was therefore adopted by the Canadian Integrated Program for Antimicrobial Resistance Surveillance.     

荧光重组酶介导等温扩增快速检测食品中克罗诺杆菌属

目的 克罗诺杆菌属为乳制品及婴幼儿配方制品中常见污染的食源性致病菌,传统的培养法检测无法满足口岸大批量食品的快速检测要求。建立快速简便的荧光重组酶介导等温扩增法(recombinase-aided amplification, RAA)检测克罗诺杆菌属,以满足口岸快速通关及监管需要。方法 根据克罗诺杆菌属ompA 基因保守区设计特异性引物、探针,通过引物两两组合结合探针筛选出扩增效率及灵敏度最佳的引物组合,优化反应温度及引物探针浓度,确定最佳反应条件。将建立的荧光RAA法应用于食品基质及实际样品检测中,同时与国标GB 4789.40-2016进行比对验证。结果 克罗诺杆菌属荧光RAA最佳反应温度为39℃,最佳引物、探针终浓度均为400nmol/L。建立的荧光RAA法特异性强,纯菌灵敏度达到3×10_² CFU/ mL。加标食品基质婴儿奶粉及婴儿米粉在月桂基硫酸盐胰蛋白胨肉汤-万古霉素(mLST)增菌只需2h,即可检测原始浓度达到3×10_^-2 CFU/ mL的克罗诺杆菌属。荧光RAA法只需5min即可观察结果,20-30min完成扩增,速度及灵敏度明显高于国标法。结论 荧光RAA法简便、快速、无需大型仪器,可用于口岸或其他场所进行克罗诺杆菌属的快速检测与监控。     

A comparison of standard cultural methods for the detection of foodborne Salmonella.

The sensitivity of the standard cultural method of the International Organization for Standardization (ISO 6579 and ISO 3565 combined) was compared to that of the Health Protection Branch (HPB) procedure for the detection of foodborne Salmonella. Of 195 foods tested, 84 (43.1%) were found to contain salmonellae by one or more cultural conditions. Of these, 75 (89.3%) and 68 (81.0%) were identified by the ISO and HPB methods, respectively. The apparent lack of agreement between both methods likely stemmed from the low indigenous numbers of salmonellae in several food homogenates, and unequal transfer of the target microorganism into homologous ISO and HPB pre-enrichment broths. The sensitivities of the commercially available Muller-Kauffmann tetrathionate broth (MKTBG43, Oxoid CM343), and a closely-related medium prepared with Oxoid CM29 tetrathionate base varied from 86.9 to 89.3%, and were deemed equivalent to that obtained with the ISO formulation of MKTBG43 (89.3%). Comparatively fewer contaminated samples were identified from selenite cystine (SC35) and selenite brilliant green (SBG35) enrichment cultures (82.1-83.3%). The high selectivity and saccharide-independent response of the bismuth sulfite agar medium warrants its consideration as a mandatory plating medium in ISO methodologies for the effective detection of typical and atypical biotypes of foodborne Salmonella spp.     

不同分离培养基检测食品中沙门氏菌效果的比较

目的基于GB 4789.4-2010沙门氏菌检验标准,比较2种沙门氏菌显色培养培养基、亚硫酸铋琼脂培养基(BS)、木糖赖氨酸脱氧胆盐琼脂培养基(XLD)等4种常用分离培养基对沙门氏菌的检测结果的影响。方法通过4种分离培养基对沙门氏菌的回收率和最低检出限的影响,添加柠檬酸杆菌和奇异变形杆菌对分离结果的影响,以及不同样品间检测效果的比较,对不同培养基的检测结果进行统计。结果 2种沙门氏菌显色培养基和XLD培养基的检测效果无差异,BS次之。结论在实际应用中,需要根据样品的污染程度选择单独或联合使用分离培养基,选择不同的前增菌培养时间,以提高检测的准确性。     

纯培养微生物荧光原位杂交技术检测的影响因素探究

为了研究纯培养微生物荧光原位杂交技术（FISH）检测的影响因素,该实验以大肠杆菌（Escherichia coli）及植物乳杆菌（Lactobacillus planetarium）为研究对象,研究不同种属、不同生长期菌株、菌体预处理方式及杂交条件对基于探针EUB338的FISH检测结果的影响。结果表明,菌体种类及菌体生长阶段均较明显影响FISH计数的准确性;确定超声分散菌体时间60 s（100 W、每30 s间歇）、溶菌酶处理60 min可较大幅度提高FISH对菌体的检出率;杂交时间和洗脱液中NaCl浓度对FISH检测结果影响小,杂交温度、杂交液中甲酰胺浓度对大肠杆菌影响小而对植物乳杆菌有一定影响。     

Direct enumeration of Escherichia coli and enteric bacteria in water,beverages and sprouts by 16S rRNA in situ hybridization

A fluorescent oligonucleotide probe complementary to a published 16S ribosomal RNA sequence ofEscherichia coli was used for in situ hybridization on membrane filters for direct enumeration of cells. The direct epifluorescent filter technique (DEFT) was modified by performing a 2-h oligonucleotide hybridization on intact cells collected on a membrane filter surface (‘oligo-DEFT’), instead of the traditional acridine orange staining. The filter was examined by epifluorescence microscopy, and digital image analysis was used for the cell enumeration. Oligo-DEFT counts were linear over the range of 10³–10⁶cells ml⁻¹and correlated with DEFT counts (r=0·99). The oligo-DEFT detection limit was approximately 1 cell ml⁻¹. Oligo-DEFT counts correlated with standard coliform counts by membrane filtration or plating on selective media for analysis of water and beverages artificially inoculated with E. coli. Heat inactivation of the cells did not destroy their reactivity in the oligo-DEFT. Natural coliform populations in aquarium water were countable in the oligo-DEFT after a 90-min incubation of the filter on nutrient agar before the hybridization step, which allowed the dormant cells to increase their rRNA content. Natural populations in bean sprouts were countable directly, without having to perform the incubation before hybridization.     

Direct application to dairy foods of a Listeria-specific oligonucleotide probe to 16S rRNA.

A potentially Listeria-specific 28 base oligonucleotide probe was designed from 16S rRNA sequence data. Using either 32P or non-radioactive (alkaline phosphatase) labels, the probe was shown to be highly specific as it hybridised to RNA extracted from all of the species of Listeria but not to any of the other bacteria tested. Both probe methods were highly sensitive and ca 10(2) cfu/ml Listeria could be detected in pure cultures. A rapid procedure for extracting RNA from milk, Camembert and cottage cheese was developed. This allowed the direct application of the probe to these foods and gave a rapid and specific method of detecting > 10(2) cfu/g or ml Listeria in these foods.     

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.     

Evaluation of viral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Norovirus GII detection

Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform.     

The development of an efficient and rapid enzyme linked fluorescent assay method for the detection of Listeria spp. from foods

Conventional isolation methods, including the Health Products and Food Branch (HPFB), Health Canada method used for the isolation and identification of Listeria species and Listeria monocytogenes from foods and environmental samples, can take a week or more to complete and are usually labor-intensive. This has led to the development of various rapid methods which attempt to generate results comparable to standard methods but in a reduced time-frame with less hands-on operation. Our previous work with rapid detection systems indicated that the recommended enrichment protocols failed to grow the Listeria to detectable levels in a reliable and consistent manner.In the present study, a novel enrichment protocol is described and consists of samples being pre-enriched in Palcam broth (incubated at 35 degrees C for 26 h), enriched in UVM 2 (30 degrees C for 26 h) and then plated and analysed by a rapid detection kit, with results being generated after 52 h of incubation.In total, 200 naturally contaminated samples were analysed by both the HPFB standard method and the Palcam method. The results showed that the Palcam method is comparable to the HPFB method. Further analysis involved a rapid detection system, which applies ELISA techniques and automation in an enzyme-linked fluorescent assay (ELFA) system. This system, referred to as the Vitek Immuno Diagnostic Assay System or VIDAS, can identify Listeria to the genus or species (L. monocytogenes) level.In this comparison, an additional 324 naturally contaminated samples were analysed by both the Palcam and ELFA methods. The sensitivity and specificity of the ELFA method were 98.1% and 97.0%, respectively, while the efficiency was 97.5%. False-negative and false-positive rates were 1.9% and 3.0%, respectively. These results show that the ELFA method (when using the Palcam method for pre- and secondary enrichment) was efficient and gave reliable results after 52 h of incubation, and met Health Canada's criteria for approval as a rapid method.     

Development of a real-time PCR-based system targeting the 16S rRNA gene sequence for rapid detection of Alicyclobacillus spp. in juice products

The thermophilic, aciduric Alicyclobacillus spp. are becoming an increasing spoilage concern in the beverage industry. Rapid methods to detect their presence in both raw materials and final products are desirable for industrial quality control. The objective of this study was to develop a real-time TaqMan-based polymerase chain reaction (PCR) system for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., for food industry applications. Two primers and a fluorogenic probe targeting 16S rRNA encoding gene sequences, capable of detecting the genus Alicyclobacillus and a few other closely related thermophiles, were developed, and the efficiency of the detection system was evaluated in both bacterial medium and juice products. Using this system, the presence of less than 100 Alicyclobacillus cells could be detected without cross-reactivity with other common food-borne bacteria.     

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%.     

食品中弓形菌16S rRNA特异性扩增检测方法的建立

针对弓形菌16SrRNA基因合成1对引物,通过对聚合酶链式反应(PCR)扩增条件的优化,建立了检测弓形菌的PCR方法。3株弓形菌标准菌株PCR产物测序结果与NCBI上公布的弓形菌16S rRNA基因序列进行比对,比对结果表明3株弓形菌测序结果与NCBI上公布的弓形菌16S rRNA基因序列同源性均在99%以上。3株弓形菌标准菌株均特异性地扩增出了长度为1202bp的片段,其他19株不同种类的菌株均无扩增产物出现。55份食品样品用Johnson-Murano肉汤增菌后用此法进行检测,其中6份样品为弓形菌阳性,阳性率为10.9%。上述实验结果表明,方法特异性强、操作简便,节省了检测时间,可用于食品中弓形菌的快速检测。     

Comparison of a microtitration plate ELISA with a standard cultural procedure for the detection of Salmonella spp. in chicken.

A rapid antibody-capture enzyme-linked immunosorbent assay (ELISA) detecting a wide range of Salmonella serotypes and employing only one culture stage was used to analyze the giblets and body cavity rinsings from frozen chickens. The results from the ELISA were compared with those obtained using a standard cultural procedure in current use in two laboratories, Norwich (N) and Ipswich (I), of the Public Health Laboratory Service (PHLS) in the UK. ELISAs were carried out on the same samples at each of two PHLS laboratories and at the Institute of Food Research with good agreement (94% and 90%). When compared with the cultural method there was 80% and 70% agreement with the ELISA with the PHLS(N) and PHLS(I) samples. The ELISA appeared to have a false-positive rate of 17% (samples from PHLS(N)) but on reculture of the "negative" samples this rate fell to 7%. The false-negative rate for the ELISA was 26% (samples from PHLS(N)) which appeared to be due to insufficient growth of the Salmonella spp. in the single cultural step employed in the ELISA rather than lack of recognition by the antibodies. The problem of false negatives with the cultural method is also discussed. These results are comparable to previously published studies relating immunoassays and the conventional procedure for Salmonella detection when analyzing similar samples. Suggestions are made as to how further increases in ELISA efficiency might be brought about.     

Rapid detection of Cronobacter spp. with a method combining impedance technology and rRNA based lateral flow assay

Cronobacter spp. is an important release test parameter for powered infant formula (PIF). An impedance method is proposed for the rapid detection of this pathogen in PIF. An impedance based method (BacTrac 4300 Microbiological Analyzer) combined with a RNA hybridisation assay (RiboFlow?) was evaluated using 23 strains in PIF samples and compared to a culture based reference method (ISO/TS 22964). The influences of competitive flora, heat and dry stress on the reliability of the impedance method were investigated. Seven different Cronobacter species were included in the evaluation, among them are strains with high susceptibility to low pH and high temperatures. Compared to the reference method, a higher sensitivity (85%) and specificity (100%) was observed using the impedance method, combined with the commercial rRNA based lateral flow test kit as a confirmation tool. The detection time was substantially shortened by using the impedance technique and RiboFlow?. Cronobacter could be detected within maximally 29h, while the reference method takes up to five days when including confirmation of positive results.