沙门氏菌快速检测板在食品检测中的应用研究

目的研究沙门氏菌快速检测板在食品检测中的应用。方法以标准菌株为测试样本,参照GB4789.4-2010验证沙门氏菌快速检测板的灵敏度、特异性、假阳性率、假阴性率、准确度,同时利用卡方检验分析阳性率显著性差异。结果沙门氏菌快速检测板最低检测限能达到10~0 CFU/m L,灵敏度为100%,不存在假阴性,特异性为92%,假阳性率为8%,准确度为95%,显著性差异卡方值卡Χ~2为0.5,均达到定性方法验证的相关指标。结论沙门氏菌检测板技术方法具有快速准确、灵敏度高、操作简单的特点,可以作为一种快速筛查方法广泛运用在食品安全微生物检测领域。     

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.     

Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples

The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for specificity, and 124 Salmonella spp. including type strains and 116 non-Salmonella strains were evaluated. All Salmonella strains tested were invA-positive and all non-salmonella strains yielded no amplification products. The melting temperature (Tm=79 degrees C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the threshold cycle (C(T)) versus copy numbers of Salmonella Enteritidis showed good linearity in broth (R2=0.994; slope=3.256) and sterilized milk (R2=0.988; slope=3.247), and the minimum levels of detection were >10(2) and >10(3) colony forming units (CFU)/ml, respectively. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated samples. Lagoon water, feed/silage, bedding soil, and bulk tank milk samples obtained from dairy farms were spiked with 10(0) to 10(5) CFU/ml of Salmonella Enteritidis. Sensitivities for detecting Salmonella in these sources were 10(3) to 10(4) CFU/ml of inoculums in broth without enrichment. Detection limits were reduced to <10 CFU/ml of inoculum in broth after 18 h enrichment. Ninety-three environmental samples including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding soil were analyzed for the presence of Salmonella by real-time PCR, results were compared with those obtained by conventional culture methods. All samples analyzed were negative for Salmonella by both real-time PCR and standard culture method. No false positive or false negative results were detected.     

PCR技术检测沙门氏菌反应条件的优化

沙门氏菌是引起细菌性食物中毒的重要病源之一,因此建立一种快速、简便、灵敏的检测方法十分重要。该研究以沙门氏菌的侵袭蛋白基因invA为靶细胞设计引物,进行PCR扩增并对PCR反应体系中引物量、模板量、dNTP、退火温度和PCR循环数等进行优化,以确定适宜的PCR反应体系和PCR扩增反应程序。     

Evaluation of motility enrichment on modified semi-solid Rappaport-Vassiladis medium (MSRV) for the detection of Salmonella in foods

The detection and identification of Salmonella spp. is still troublesome and time consuming to the food industry. Employing the modified semi-solid Rappaport-Vassiliadis medium (MSRV), presumptive results for Salmonella can be obtained in 48 h, representing an interesting alternative to the standard methods. The specificity and sensitivity of the MSRV method were evaluated in this research. The efficiency of this method was also compared with the methodology recommended by the US Food and Drug Administration (FDA) using bismuth sulfite agar, XLT4 agar and Rambach agar. A total of 146 food samples comprised of 41 chicken thighs, 35 Brazilian fresh pork sausages, 35 samples of cocoa powder and/or granulated cocoa and 35 samples of grated fresh coconut, were examined. Overall, the rapid method (direct + indirect) and the standard culture detected 96.1% and 84.6% of the positive samples, respectively. No Salmonella was detected in the coconut or cocoa samples by any of the methods. Eighteen (43.9%) chicken thigh samples were contaminated with the microorganism. The rapid method (direct + indirect) and the standard culture detected 94.4% and 88.9% of these, respectively. Salmonella was detected in eight (22.8%) fresh pork sausage samples. The MSRV method detected Salmonella in all eight samples, while the standard gave positive results in six (75%). When compared with the standard method, the indirect method showed 86.4% sensitivity and 96.8% specificity, while the direct MSRV showed a sensitivity of 71.4% and specificity of 99.2%. Combined, both MSRV methods showed 95.5% sensitivity and 96.8% specificity. The MSRV medium also reduces the time necessary for the isolation of Salmonella from foods.     

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.     

PCR Detection and Microbiological Isolation of Salmonella spp. from Fresh Beef and Cantaloupes

ABSTRACT:  Species belonging to the genus Salmonella are an important cause of enteric fevers, gastroenteritis, and septicemia, and the pathogens are commonly transmitted through contaminated food. In this study, polymerase chain reaction (PCR) amplification of a 287-bp region of the invA gene was compared to a microbiological technique to determine the presence of Salmonella in retail beef and in cantaloupe rinse samples. Both methods showed the same level of sensitivity, detecting 1 CFU/25 g of meat after enrichment for 24 h at 42 °C. The presence of Salmonella was determined in 50 commercial top sirloin beef samples that were not artificially inoculated. Three samples were positive by the microbiological method, and these samples and an additional sample were positive by the PCR. Both methods were also used to test surface rinses of cantaloupes collected from 4 farms in Nayarit, Mexico. Salmonella was detected by the microbiological method in 9 of 20 samples (45%), whereas the pathogen was detected by the PCR in 11 samples (55%). This study demonstrates the utility of the PCR targeting the invA gene to determine the presence of Salmonella spp. in beef and cantaloupe samples.     

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.     

Evaluation of an immunomagnetic separation-real-time PCR assay for the rapid detection of Salmonella in meat

The aim of this study was the comparison of an immunomagnetic separation (IMS)-real-time PCR assay for the detection of Salmonella with the cultural reference method according to and 35 of the German Law on Food and Commodities (LMBG, L 00.00.20:1998). The IMS-real-time PCR assay includes a nonselective preenrichment step, an IMS, DNA extraction, as well as DNA purification followed by hybridization probe-based real-time PCR analysis. An accurate comparability was achieved, because both methods analyzed the same preenrichment. The evaluation was carried out using both artificially and naturally contaminated meat samples. The IMS-real-time PCR assay provides a result after 12 to 13 h. Compared with the reference method and regarding artificially contaminated meat samples, the IMS-real-time PCR assay achieved a specificity of 80% (false-positive rate of 20%) and a sensitivity of 100% (false-negative rate of 0%). The relative accuracy was 94%. The detection limit of both methods was 10 CFU/25 g. The concordance index kappa, which defines the statistical accordance, was 0.85 and indicated the agreement of both methods on statistical criteria. Compared to the reference method and analyzing naturally contaminated meat samples (n = 491), the IMS-real-time PCR assay showed a specificity of 99.3% (false-positive rate of 0.7%) and a sensitivity of 83.7% (false-negative rate of 16.3%). The relative accuracy was 98%. The concordance index kappa had a value of 0.87 and highlighted the statistical agreement of both methods. In conclusion, the IMS-real-time PCR assay is suitable as specific, sensitive, and rapid screening method for the detection of Salmonella from meat.     

Evaluation of a 24-hour bioluminescent enzyme immunoassay for the rapid detection of Salmonella in chicken carcass rinses

A bioluminescent enzyme immunoassay (BEIA), using Salmonella-specific monoclonal antibody M183 for capture and biotinylated monoclonal antibody M183 for detection, was developed with InteLite AB streptavidin-biotinylated firefly luciferase complex as a reporter. Salmonella cultures were preenriched in buffered peptone water with shaking for 6 h at 37 degrees C and then selectively enriched in Muller-Kauffmann tetrathionate (MKTT) broth and modified semisolid Rappaport-Vassiliadis (MSRV) medium for 16 h at 42 degrees C. After enrichment, the total test time for the BEIA was 1.5 h. The analytical sensitivity of the BEIA ranged from 6.0 x 10(2) CFU/ml to 1.2 x 10(5) CFU/ml in MKTT and from 1.4 x 10(5) to 2.3 x 10(6) CFU/ml in MSRV using six Salmonella serovars prevalent in Canada. With enrichment cultures, the BEIA detected 1 CFU of Salmonella Typhimurium and Salmonella Enteritidis in 25 ml of chicken rinses. Representative strains of 10 Salmonella serovars were detected, and cross-reactivity was not observed with 25 non-Salmonella foodborne bacteria. The BEIA performance was assessed by testing 420 poultry samples, which were analyzed in parallel with the standard MSRV culture method. The BEIA detected 117 (27.88%) Salmonella-positive samples, whereas the standard MSRV culture method detected 124 (29.5%). The BEIA had a sensitivity of 64.5% and a specificity of 87.5% compared to the standard MSRV culture method. However, similar specificities and sensitivities were obtained when the standard MSRV culture method was compared to the BEIA (sensitivity = 68.4% and specificity = 85.5%). Neither method detected 100% of the Salmonella found in the samples tested, and statistical analyses indicated no significant difference between the two methods. In summary, the BEIA offers another alternative for the detection of Salmonella, with the additional advantage of providing a 24-h test for detecting Salmonella in chicken carcass rinses. The results obtained in this research indicate that tests are still needed for the isolation and detection of Salmonella that will establish the true prevalence of Salmonella in chicken samples.     

PCR技术检测动物源性食品中沙门菌的应用研究

为快速准确检测动物源性食品中的沙门菌,将建立并优化的沙门菌PCR检测试剂盒应用于动物源性食品的检测,并于国标法进行了比较.对上海地区238份鸡蛋、685份原料牛奶、283份猪肉、314份牛肉和58份虾仁样品的沙门菌检测表明,PCR法的敏感性和特异性均为100%,与国标法的符合率亦为100%,且检测时间仅为2 d,较国标法大为缩短.该方法可用于动物源性食品中沙门菌的检测,并具有快速、特异、敏感等优点。     

A new rapid automated method for the detection of Listeria from environmental swabs and sponges

Many food and meat processors test environmental swabs and sponges to confirm the absence of Listeria spp. Spectral pattern changes in a liquid growth medium, resulting from esculin hydrolysis by Listeria in contaminated swabs and sponges, were automatically monitored by the BioSys instrument in a semifluid layer (SFL). The blackening of SFL in modified MOX broth resulted in sharply declining curves, which were easily detected by the instrument. The instrument detected all nine strains of Listeria monocytogenes tested. None of the gram negative organisms (Proteus, Escherichia coli, Pseudomonas, Citrobacter and Yersinia) were detected by the system, nor were most gram positive organisms, including Bacillus, Streptococcus, and Lactobacillus strains, Staphylococcus aureus, Enterococcus faecium and E. faecalis, which hydrolyze esculin, produced black colonies on PALCAM and Oxford media and were also detected in the system. A total of 122 sponges and swabs collected at food processing plants were evaluated by this method. Of these, 99 were negative, and 11 were positive. L. innocua was the dominant Listeria species in these environmental samples. Good correlation was obtained between numbers of Listeria and detection times of esculin hydrolysis: 1000 CFU/swab were detected in 10-13 h, whereas 1-10 CFU/swab were detected in less than 22 h. The total assay time was 26 h.     

Multiplex PCR assay and lyophilization for detection of Salmonella spp., Staphylococcus aureus and Bacillus cereus in pork products

Food Science and Biotechnology - Multiplex PCR (m-PCR) has the potential for more rapid detection of pathogens compared to simple PCR through the simultaneous amplification of multiple gene targets...     

Development of a rapid FLISA detection of Salmonella spp. based on CdTe/ZnS quantum dots

Salmonella pathogen is old and difficult to control in food safety. The study was aimed to develop a sensitive and rapid CdTe/ZnS quantum dots (QDs) mediated fluorescence-linked immunosorbent assay (FLISA) of Salmonella spp. The optical properties and structure of the CdTe/ZnS QDs were characterized by UV–Vis absorption spectroscopy and transmission electron microscopy. The emission peak wavelength is 575 nm. The CdTe/ZnS QDs-labeled secondary antibody (goat anti-rabbit IgG) was formed by biotin-avidin system. The detection limits of S. Enteritidis and artificial simulated samples are 1.0 × 10³ CFU/ml and 1.78 × 10³ CFU/ml, respectively. The study of FLISA stability shows that the antibody with QDs had very good activity when it was stored in the dark at 4°C for at least 35 days. The FLISA provides a new and easy-operated method for the rapid detection of Salmonella spp. and has broad application in food safety.     

Prevalence and risk factors for Salmonella and Campylobacter spp. carcass contamination in turkeys slaughtered in Quebec, Canada

An observational study was conducted to estimate prevalence and risk factors for carcass contamination by Salmonella and Campylobacter spp. in 60 lots of turkey slaughtered over 10 months in the province of Quebec, Canada. Carcass contamination was evaluated by the carcass rinse technique for about 30 birds per lot. Exposure to potential risk factors was evaluated with questionnaires, meteorological data, and cecal cultures. Multivariable binomial negative regression models were used for risk factor analysis. Prevalence of Salmonella-positive carcasses was 31.2% (95% confidence interval, 22.8 to 39.5%). Variables positively associated (P < or = 0.05) with the proportion of lot-positive carcasses were > or =0.5% of carcass condemnation due to various pathologies, cecal samples positive for Salmonella, low wind speed during transportation, closure of lateral curtains of truck during transportation, and slaughtering on a weekday other than Monday. When only Salmonella-positive cecal culture lots were considered, the proportion of carcasses positive for Salmonella was significantly higher in lots exposed to a >5 degrees C outside temperature variation during transportation, slaughtered on a weekday other than Monday, and in which > or = 4% of carcasses had visible contamination. Prevalence of Campylobacter-positive carcasses was 36.9% (95% confidence interval, 27.6 to 46.3%). The proportion of positive carcasses was significantly higher in lots with Campylobacter-positive cecal cultures and lots undergoing > or =2 h of transit to slaughterhouse. For lots with Campylobacter-positive cecal cultures, variables significantly associated with an increased incidence of carcass contamination were de4% of carcasses with visible contamination, crating for > or =8 h before slaughtering, and no antimicrobials used during rearing.     

速冻食品中沙门氏菌和金黄色葡萄球菌多重PCR检测方法的建立与应用

为实现速冻食品中沙门氏菌(Salmonella spp.)和金黄色葡萄球菌(Staphylococcus aureus)的多重聚合酶链式反应(PCR)检测,首先优化多重PCR扩增的反应条件,比较基因组DNA提取方法,结果表明：退火温度采用60℃、各引物浓度200nmol/L及扩增循环35次,本多重PCR检测技术可以有效地将沙门氏菌和金黄色葡萄球菌同时检出,检测特异性为100%。3种DNA提取方法中试剂盒法纯度最高,检出限分别是31、26DNA copies/reaction。经过在人工污染致病菌的速冻水饺中应用试验后,该多重PCR方法经过4h的增菌培养即可从速冻水饺中同时检测出起始菌落数低至100CFU/g的沙门氏菌和金黄色葡萄球菌。     

Salmonella in food surveillance: PCR,immunoassays, and other rapid detection and quantification methods

Foodborne diseases caused by Salmonella have always been a significant health burden to many countries. Recent epidemiological data have indicated that Salmonella was the most common bacterial etiologic agent in food poisoning outbreaks both in the United States and in Asian regions like Hong Kong. In the past, labor-intensive traditional standard culture methods with long turnaround time have always been employed by many laboratories of public health services for the detection of Salmonella in Food Surveillance Programmes. To cope with the enormous volume of sample received for Salmonella detection, recent advances in nucleic acid- and immunoassay-based methods, and the subsequent commercialization and automation of the technologies have provided more rapid, specific, and productive alternatives for routine applications in testing laboratories. Fluorogenic or real-time PCR methods are able to generate results in a day, whereas immunoassay-based methods can produce negative results in 1–3 days. Some of these rapid methods have already been validated and accepted by international authorities as standard methods and have become increasingly popular among testing laboratories.     

基于免疫磁分离-双重荧光定量PCR的鲜猪肉中沙门氏菌和志贺氏菌的检测

目的使用免疫磁分离-双重荧光定量PCR方法实现对鲜猪肉中金黄色葡萄球菌和志贺氏菌的同时、快速检测。方法在37℃的条件下,利用特异性免疫磁球从250 m L循环体系中快速、有效地捕获目标菌。再通过特异性的引物与探针,对沙门氏菌和志贺氏菌进行双重荧光定量PCR检测。结果本研究方法针对鲜猪肉中沙门氏菌和志贺氏菌的检测限分别达到3.4 cfu/g和9.4 cfu/g。方法总体灵敏度、特异性和准确度均达到100%。此外,通过对30组实际样品的检测,该方法与传统标准方法的结果保持一致。结论本研究建立的免疫磁分离-双重荧光定量PCR方法比国标方法更快速和高效,适用于鲜猪肉中沙门氏菌和志贺氏菌的快速检测。     

Prevalence and risk factors for Salmonella and Campylobacter spp. carcass contamination in broiler chickens slaughtered in Quebec, Canada

An observational study was conducted to estimate prevalence and risk factors for Salmonella and Campylobacter spp. carcass contamination in broiler chickens. Eighty-two lots were sampled in four slaughterhouses located in the province of Québec, Canada, over a 10-month period. Carcass contamination was evaluated by the carcass rinse technique for about 30 birds per lot. Exposure to potential risk factors was evaluated based on data from questionnaires, meteorology, and cecal cultures. Multivariable binomial negative regression models were used for risk factor analysis at the lot level. The prevalence of Salmonella-positive carcasses was 21.2% (95% confidence interval: 15.7 to 26.7%). Significant risk factors (P < 0.05) associated with a higher proportion of positive carcasses within lots were Salmonella-positive cecal culture, low rainfall during transportation to the slaughterhouse, temperature of > or = 0 degree C during transportation to the slaughterhouse, and a > or = 4-h waiting period in shipping crates before slaughtering. The prevalence of Campylobacter-positive carcasses was 35.8% (95% confidence interval: 27.1 to 44.5%). Lots containing birds with Campylobacter-positive cecal culture results, lots of birds that were slaughtered at the end of the week, and lots with at least 20% of birds with digestive contents detected in the jejunum at time of slaughtering had a significantly higher proportion (P < 0.05) of contaminated carcasses. These results support the importance of preharvest control measures implemented during rearing to reduce contamination of the final product. Weather during transportation to slaughter and the day of the week that birds were slaughtered also were associated with carcass contamination; further studies are needed to determine the underlying mechanisms by which these factors influence carcass contamination.     

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.