PCR detection of Salmonella spp. using primers targeting the quorum sensing gene sdiA

Bacteria communicate with one another and with their host using chemical signalling molecules. This phenomenon is generally described as quorum sensing. A set of primers for PCR detection of Salmonella spp. has been designed using as target the sdiA gene which encodes a signal receptor of the LuxR family. The PCR product (274 bp) was confirmed by sequencing. A number of 81 non-Salmonella strains (representing 24 different species) were tested and gave negative results, while a total of 101 different serotypes of Salmonella (155 strains) tested positive for the presence of the sdiA gene. The sensitivity and specificity of the sdiA-based PCR assay were also checked in artificially contaminated human faecal samples. In this study, we demonstrate that quorum sensing genes can be successfully exploited as diagnostic markers.     

Importance of pre-enrichment media for isolation of Salmonella spp. from swine and poultry

The performance of two new (1-day) culture methods, Salmonella Enrichment Broth (SEB) and Revive, and an alternative pre-enrichment broth, designated Universal pre-enrichment broth (UB), was compared to the internationally accepted buffered peptone water (BPW). The study was directed towards detection of Salmonella in 100 faecal samples from porcine and 100 neck-skin samples from poultry. The sensitivity (number of positive cases per method among all the positive cases) of the conventional pre-enrichment in BPW was found to be 0.77 for swine and 0.66 for poultry samples, while a combination of the BPW method with parallel pre-enrichment of the same sample in UB resulted in high sensitivity for swine (0.92) and poultry (0.95) samples. A 2-h pre-enrichment in the non-selective Revive, followed by overnight enrichment in selective broth, resulted in a low sensitivity, particularly for the neck-skin samples (0.16, P=0.001). The SEB method in the porcine samples resulted in a sensitivity (0.71) comparable to the standard method (P=0.31). In conclusion, additional pre-enrichment of samples in UB may substantially increase the culture sensitivity. During routine screening of large numbers of samples, it may be advantageous to use SEB rather than standard culturing.     

2005-2013年肠道致病菌中志贺菌和沙门菌的分布及检测方法经验总结

目的分析杭州市第三人民医院2005-2013年肠道致病菌中志贺菌和沙门菌的分布并对检测方法进行经验总结。方法采用定位显色培养基和营养琼脂初筛疑似志贺菌和沙门菌,并参考该院历年菌种分离情况进行血清凝集。采用EXCEL统计处理数据,χ2检验比较采用定位显色培养基和营养琼脂平板之前和之后,粪便培养志贺菌和沙门菌的分离率之间差异的显著性。结果采用定位显色培养基和营养琼脂平板初筛疑似菌株后,志贺菌和沙门菌的分离率差异存在统计学意义。结论采用定位显色培养基和营养琼脂初筛疑似菌株,并对本院历年菌种分离情况进行总结,对改善志贺菌和沙门菌的分离率,提升检验科微生物室的工作效率有较大帮助。     

Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach

AIMS: To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR). METHODS AND RESULTS: Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 10(1)-10(6) CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen. CONCLUSIONS: When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 10(3) and 10(1) CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products.     

食品中沙门氏菌分子检测靶点的筛选与评价

[目的]发掘新的沙门氏菌分子检测靶点,筛选检测性能优秀的引物.[方法]利用BLAST程序比较沙门氏菌属内基因组DNA序列的同源性以及沙门氏菌与非沙门氏菌基因组DNA序列之间的特异性,发掘出100多个检测沙门氏菌属的特异性片段,并从中随机挑选出15个片段作为候选靶点,一共设计了27对引物(FS1～FS27),对它们的特异性、灵敏度加以评价,从中筛选检测性能最好的引物.[结果]在27对引物中,检测性能最优的引物为FS23,采用该引物对供试菌株的相应检测靶点进行PCR扩增,44株沙门氏菌都能扩增到一条492 bp特异性片段,而22株非沙门氏菌则不能扩增出这一特异性片段.以FS23为引物建立PCR方法检测猪霍乱沙门氏菌基因组DNA的灵敏度为11.9 fg/μL,细菌纯培养物灵敏度为4.9×102cfu/mL;用猪霍乱沙门氏菌人工污染牛奶样品,如果接种起始菌量为100 cfu/25 mL时,只需要增菌5 h,采用上述方法即能检测出沙门氏菌.[结论]引物FS23对应的基因序列是一个性能优良的新分子检测靶点,具备很高的特异性和灵敏性,能够广泛应用于食品中沙门氏菌的快速检测.     

Real-time PCR method for Salmonella spp. targeting the stn gene

AIM: To develop a real-time PCR assay for Salmonella spp. targeting the stn gene. METHODS AND RESULTS: The presence of stn in the Salmonella bongori genome was found by a BLAST with Salmonella enterica stn sequence. Manual alignment of stn sequences showed that Salm. bongori had 88% sequence identity with Salm. enterica. Two primers (stnL-433 and stnR-561) and a probe (stnP-452) were designed to target conserved regions in stn and meet the requirements of a 5'-nuclease assay. The primers and probe were evaluated against 353 isolates, including 255 Salm. enterica representing 158 serotypes, 14 Salm. bongori representing 12 serotypes and 84 non-Salmonella representing 56 species from 31 genera. All isolates were correctly identified, with the exception of three isolates of Citrobacter amalonaticus, which gave false positives. The limit of detection with cultured Salmonella was 3 CFU per reaction. CONCLUSIONS: The stn real-time PCR method had 100% inclusivity, 96.4% exclusivity and a level of detection of 3 CFU per reaction for cultured Salmonella spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that stn is present in Salm. bongori and is a valid target for both species of Salmonella. The Salmonella s tn real-time PCR is a useful method for identifying Salmonella spp.     

A rapid monitoring assay for the detection of Salmonella spp. and Salmonella Senftenberg strain W775 in composts

AIMS: The composting process needs to be validated for its hygienic status in order to ensure that it is free of pathogens. Generally, this is evaluated through temperature monitoring, or additionally through active inoculation and monitoring of indicator organisms. We aimed to develop a monitoring method for the heat-resistant indicator organism Salmonella enterica ssp. enterica serovar Senftenberg strain W775 for detection in composting biowastes. METHODS AND RESULTS: The method development is comprised of: (i) optimization of molecular detection of bacteria belonging to the genus Salmonella; (ii) identification of a DNA marker for Salmonella strain W775; and (iii) development of a multiplex polymerase chain reaction (PCR)-based on both DNA markers. Subsequently, Salmonella strain W775 was inoculated and monitored during composting of biowastes in an industrial composting facility. CONCLUSIONS: A highly sensitive and specific detection of viable cells was obtained by enriching the compost sample prior to multiplex PCR analysis. Complete inactivation of Salmonella strain W775 was obtained within 4 days in an industrial composting facility at temperatures ranging between 41 and 57 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a monitoring method for the simultaneous detection of naturally occurring Salmonella strains and artificially introduced Salmonella strain W775 in composting biowastes that can be applied in routine analysis.     

Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.     

Evaluation of selective and non-selective enrichment PCR procedures for Salmonella detection

AIMS: To compare PCR combined with enrichment media with the standard microbiological techniques (SMT) and to determine the most sensitive method for the detection of Salmonella and the identification of Salm. typhimurium (ST), Salm. enteritidis (SE), Salm. gallinarum (SG) and Salm. pullorum (SP). METHODS AND RESULTS: We analysed 87 samples from poultry using PCR and SMT, PCR being performed from non-selective (NS) and Rappaport-Vassiliadis (RV) media. PCR-NS was less sensitive than PCR-RV and SMT for the detection and identification of Salmonella. PCR-RV detected more positive samples of Salmonella sp. than SMT but both these methods showed similar sensitivity regarding the identification of Salmonella serovars. CONCLUSIONS: PCR-RV was more sensitive and decreased the time necessary to detect and identify Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-RV is a powerful tool for the rapid and accurate detection and identification of Salmonella and can be implemented in diagnostic and food analysis laboratories.     

Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction

Aims: A new real‐time polymerase chain reaction‐based method was developed for the detection of Salmonella enterica in food. Methods and Results: The method consisted of a novel two‐step enrichment involving overnight incubation in buffered peptone water and a 5‐h subculture in Rappaport–Vassiliadis medium, lysis of bacterial cells and a Salmonella‐specific 5′‐nuclease real‐time PCR with an exogenous internal amplification control. Because a two‐step enrichment was used, the detection limit for dead S. enterica cells in artificially contaminated ice cream and salami samples was high at 10⁷ CFU (25 g)^?1, eliminating potential false‐positive results. When the method was evaluated with a range of 100 naturally contaminated food samples, three positive samples were detected by both the real‐time PCR‐based method and by the standard microbiological method, according to EN ISO 6579. When the real‐time PCR‐based method was evaluated alongside the standard microbiological method according to EN ISO 6579 with 36 food samples artificially contaminated at a level of 10⁰ CFU (25 g)^?1, identical results were obtained from both methods. Conclusions: The real‐time PCR‐based method involving a two‐step enrichment produced equivalent results to EN ISO 6579 on the day after sample receipt. Significance and Impact of the Study: The developed method is suitable for rapid detection of S. enterica in food.     

Automated detection of Salmonella spp. in foods.

An automated method to detect salmonellae in foods was developed and tested in food samples intentionally contaminated with the test organisms. Liquid eggs, shell eggs, dry eggs, skim milk and chicken were spiked with Salmonella enteritidis, S. typhimurium or S. newport to yield 2 to 25 CFU per 25 g or ml of sample. Following pre-enrichment in universal pre-enrichment broth at 42 degrees C for 6 h (eggs and milk) or 16 h (chicken), Salmonella cells were captured by immunomagnetic beads coated with Salmonella antibody (Vicam, Watertown, MA). The beads were transferred to selective liquid media containing carbohydrate (dulcitol or xylose), amino acid (lysine or ornithine), and H2S indicator, and incubated at 42 degrees C in the BioSys instrument (MicroSys, Ann Arbor, MI). Salmonella positive samples were identified by black discoloration of the media during incubation, while negative samples remained colorless. These color changes were recorded by the instrument. All the artificially contaminated samples tested positive within 15-18 h, while control samples remained negative during 24 h incubation. The results agreed with standard identification procedures. A total of 24 h was required to detect 2 to 25 CFU of the pathogen in 25 g or ml of eggs and milk, and up to 36 h in chicken, compared to 72 h in the standard methods.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe.

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Design of new primers that can detect Salmonella without decrease of detection threshold in the presence of contaminant organisms

Novel PCR primer sets for the detection of Salmonella were designed based on the oriC sequence information of various enteric bacteria, which are potential contaminants in commercial liquid egg samples. Using the PCR primers, the selective detection of Salmonella in the presence of large excess number of salmonella-like strains were achieved without the reduction of its sensitivity. The application of these primer sets for the detection of Salmonella from commercial liquid egg was also demonstrated.     

Rapid and simple detection of the invA gene in Salmonella spp. by isothermal target and probe amplification (iTPA)

Aims: Salmonella spp. are an important cause of food‐borne infections throughout world, and the availability of rapid and simple detection techniques is critical for the food industry. Salmonella enterica serovars Enteritidis and Typhimurium cause the majority of human gastroenteritis infections, and there are a reported 40 000 cases of salmonellosis in the United States each year. Methods and Results: A novel rapid and simple isothermal target and probe amplification (iTPA) assay that rapidly amplifies target DNA (Salmonella invA gene) using a FRET‐based signal probe in an isothermal environment was developed for detection Salmonella spp. in pre‐enriched food samples. The assay was able to specifically detect all of 10 Salmonella spp. strains without detecting 40 non‐Salmonella strains. The detection limit was 4 × 10¹ CFU per assay. The iTPA assay detected at an initial inoculum level of <10 CFU in the pre‐enriched food samples (egg yolk, chicken breast and peanut butter). Conclusions: This detection system requires only a water bath and a fluorometer and has great potential for use as a hand‐held device or point‐of‐care‐testing diagnostics. The iTPA assay is sensitive and specific and has potential for rapid screening of Salmonella spp. by food industry.