Detection of Salmonella spp. in oysters by PCR.

PCR DNA amplification of a region of the himA gene of Salmonella typhimurium specifically detected Salmonella spp. In oysters, 1 to 10 cells of Salmonella spp. were rapidly detected by the PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of viable Salmonella spp.     

Rapid and specific detection of Salmonella spp. in animal feed samples by PCR after culture enrichment

A PCR procedure has been developed for routine analysis of viable Salmonella spp. in feed samples. The objective was to develop a simple PCR-compatible enrichment procedure to enable DNA amplification without any sample pretreatment such as DNA extraction or cell lysis. PCR inhibition by 14 different feed samples and natural background flora was circumvented by the use of the DNA polymerase Tth. This DNA polymerase was found to exhibit a high level of resistance to PCR inhibitors present in these feed samples compared to DyNAzyme II, FastStart Taq, Platinum Taq, Pwo, rTth, Taq, and Tfl. The specificity of the Tth assay was confirmed by testing 101 Salmonella and 43 non-Salmonella strains isolated from feed and food samples. A sample preparation method based on culture enrichment in buffered peptone water and DNA amplification with Tth DNA polymerase was developed. The probability of detecting small numbers of salmonellae in feed, in the presence of natural background flora, was accurately determined and found to follow a logistic regression model. From this model, the probability of detecting 1 CFU per 25 g of feed in artificially contaminated soy samples was calculated and found to be 0.81. The PCR protocol was evaluated on 155 naturally contaminated feed samples and compared to an established culture-based method, NMKL-71. Eight percent of the samples were positive by PCR, compared with 3% with the conventional method. The reasons for the differences in sensitivity are discussed. Use of this method in the routine analysis of animal feed samples would improve safety in the food chain.     

Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

Background

Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.

Methods

Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.

Results

An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens.

Conclusions

The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.     

Specific Oligonucleotide Primers for Detection of Lecithinase-Positive Bacillus spp. by PCR

Volume 61, no. 1, p. 100, Table 3: in columns 7 and 8, row 3, "-" should read "+." [This corrects the article on p. 98 in vol. 61.].     

畜禽肉沙门氏菌和大肠杆菌O157多重PCR检测研究

沙门氏菌和大肠杆菌O157都是目前世界公认引起食源性疾病的重要致病菌.本研究针对致病茵传统检测方法耗时长、过程繁琐的缺点,建立了同时检测畜禽肉及其制品中沙门氏菌和大肠杆菌O157的多重PCR分子检测方法.结果表明:分别针对沙门氏茵侵袭基因invA、大肠杆菌O157抗原基因rfbE建立的多重PCR方法可简便、快速、灵敏地实现对沙门氏菌和大肠杆菌O157的同时检测,整个过程在9h～10h内完成,人工污染猪肉检测限分别达到2.4×102cfu/mL(沙门氏菌)和2.2×102 cfu/mL(大肠杆菌O157);为食源性致病菌的检测提供了理想手段,有良好的应用前景.     

用环介导等温扩增技术快速检测粪便样本中的沙门菌

目的:建立快速检测粪便样本中的沙门菌的环介导等温扩增技术(LAMP),并着重在灵敏度和特异性方面对此方法进行评价。方法:利用LAMP针对沙门菌特定基因invA(靶基因)设计的6条特异引物,通过引物特异性识别特定基因invA上的8个独立区域来快速检测沙门菌;LAMP反应过程中会产生白色沉淀焦磷酸镁,故可以通过监测浊度来判定反应结果。结果:实时浊度仪监测反应结果表明,LAMP反应在60~65℃等温条件下50 min内完成;如果在反应前添加羟基萘酚兰,蓝色阳性结果很明显区别于紫色阴性结果;LAMP的最低检出限为6.97 pg/μL,PCR为69.7pg/μL,LAMP方法的检测灵敏度是PCR的10倍,且具有良好的特异性。结论:LAMP方法用于快速检测沙门菌,具有检测过程简单、实验装置简便、反应结果肉眼可辨、灵敏度高、特异性强的特点,对非沙门菌菌株的结果呈阴性,表明引物设计有很好的特异性。对粪便样本进行检测,发现具有同样的敏感性和特异性。这表明LAMP法是潜在的和有价值的在粪便样本中直接检测沙门菌的方法,具有快速、简便、低成本的特点。LAMP法适用于快速临床诊断。     

基于特异性PCR和荧光检测技术快速鉴定艾纳香

为建立快速准确的艾纳香分子鉴定方法。采取筛选艾纳香及其混伪品基因组DNA的提取方法,针对艾纳香特异性位点设计引物,优化PCR扩增条件,荧光检测扩增产物。结果表明碱裂解法更适于艾纳香基因组DNA的提取;叶绿体基因（tDNA）特异引物能特异性扩增艾纳香DNA,其扩增产物荧光检测呈绿色,混伪品无反应发生。试验结果显示该法简化了分子鉴定过程,省时节力,且结果准确可靠,可作为艾纳香植物和药材的鉴定方法。     

沙门菌、志贺菌、副溶血性弧菌多重PCR检测方法的研究

建立快速检测沙门菌、志贺菌和副溶血性弧菌的多重PCR方法[1-4].根据沙门菌hilA基因、志贺菌ipaH基因及副溶血性弧菌TDH基因设计特异性PCR引物[5-6],被检样品经4 h振荡培养后金属浴裂解制备DNA模板,使用全自动毛细管电泳核酸检测系统分析PCR扩增产物.在580、423和245 bp处分别出现预期的特异性DNA条带,且无非特异扩增条带出现.敏感性试验显示沙门菌在模拟标本中的检测灵敏度为101-2cfu/mL、志贺菌为101cfu/mL、副溶血性弧菌为102cfu/mL.该方法操作方便、分析时间短、特异性和灵敏度高,可用于公共卫生突发事件食源性病原菌的快速检测.     

Rapid Detection of Salmonella spp. by Using Felix-O1 Bacteriophage and High-Performance Liquid Chromatography

A method is described whereby the presence of Salmonella spp. can be detected within 8 to 24 h of sample collection. The method depends upon the interaction of Salmonella spp. with the Salmonella-specific Felix-O1 bacteriophage. This interaction results in an increase in concentration of the bacteriophage which is detected by high-performance liquid chromatographic techniques.     

沙门菌、大肠杆菌和金黄色葡萄球菌的多重PCR检测

根据沙门菌invA基因、大肠杆菌phoA基因和金黄色葡萄球菌nuc基因序列,设计3对特异性引物进行多重PCR并对反应条件进行优化。结果表明3对引物能特异地扩增出284bp、622bp、484bp的目的条带;最佳反应条件为沙门菌、大肠杆菌、金黄色葡萄球菌的引物浓度分别为40nmol／L、40nmol／L、80nmol／L,Mg^2＋浓度2．4mmol／L,dNTP浓度2001μmol／L,Taq DNA聚合酶1.5u,退火温度55．0℃-57．4℃之间;在此条件下多重PCR同时检测DNA的敏感性分别是10．2pg、10．2pg、102．0pg,检测时间4h。建立的多重PCR是一种敏感、特异、准确、快速的方法,为同时检测食品中沙门菌、大肠杆菌和金黄色葡萄球菌奠定了基础。     

PCR技术在快速检测沙门氏菌中的应用

本文总结了PCR检测沙门氏菌过程中被检目标基因的选择、引物的特异性以及国内外检测沙门氏菌的一些实例,分析了PCR检测沙门氏菌在引物选择上存在的问题,简述了在传统PCR技术基础上发展起来的检测沙门氏菌新方法。     

Detection of Salmonella spp. and Listeria monocytogenes in Suspended Organic Waste by Nucleic Acid Extraction and PCR

A nucleic acid-based method for the detection of the bacterial pathogens Salmonella spp. and Listeria monocytogenes in biological waste was developed. The detection limits were less than 10 cells per ml of biological waste. The method does not include a phenol extraction step and can be easily performed in 1 to 2 days.     

Detection of Salmonella spp. and Listeria monocytogenes in suspended organic waste by nucleic acid extraction and PCR

A nucleic acid-based method for the detection of the bacterial pathogens Salmonella spp. and Listeria monocytogenes in biological waste was developed. The detection limits were less than 10 cells per ml of biological waste. The method does not include a phenol extraction step and can be easily performed in 1 to 2 days.     

Simple and Rapid Detection of Salmonella by Direct PCR Amplification of Gene fimW

This study established a simple method of specifically detecting Salmonella species by amplifying fimW gene, which was involved in regulating Salmonella type I fimbriae expression. A pair of primers was designed to target and discriminate the 68 Salmonella strains of 23 Salmonella serovars available to us from 12 non-Salmonella strains of five different kinds of bacteria by polymerase chain reaction (PCR) amplification. Results showed that specific DNA fragment with an expected size of 477 bp was successfully amplified from all Salmonella serovars, while no target band was detected in non-Salmonella species. The sensitivity of this PCR-amplifying system reached to 1 pg DNA chromosome and 10² cfu of Salmonella enteritis strain CMCC(B) 50336. The above results demonstrated the method as a simple, sensitive, and specific way for Salmonella detection.     

A Real-Time PCR Assay for the Detection of Campylobacter jejuni in Foods after Enrichment Culture

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37°C for 24 h, followed by 42°C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.     

The Use of Enrichment Serology for Salmonella Detection in Human Foods and Animal Feeds

S ummary . Samples (2208) of food raw materials and products were examined for the presence of salmonellae by use of conventional salmonella detection procedures and the enrichment serology (ES) techniques described by Sperber & Deibel (1969); 348 samples were positive for salmonellae by the conventional procedures. Using the ES technique with a 24 h elective enrichment step, 93–98% of samples positive by the conventional procedures were also positive by the ES technique. Selective enrichment of food samples using tetrathionate broth containing novobiocin, incubated at 41°, led to the best recovery of salmonellae by both the conventional and ES techniques.     

Specific detection of Salmonella spp. by multiplex polymerase chain reaction.

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.     

Specific oligonucleotide primers for detection of lecithinase-positive Bacillus spp. by PCR.

An assay based on the PCR has been developed to facilitate detection and identification of Bacillus cereus in foods. Three primers for the PCR have been designed within the sequence for cereolysin AB, a cytolytic determinant that encodes lecithin-hydrolyzing and hemolytic activities of B. cereus. With the PCR and hybridization, the specificity of the primers was tested with 39 isolates of the B. cereus group, with 17 other Bacillus spp., and with 21 non-Bacillus strains. Results demonstrate a high specificity of the three oligonucleotides for isolates of the B. cereus group. With a combined PCR-hybridization assay, the detection limit for B. cereus in artificially contaminated milk was 1 CFU/ml of milk.     

Rapid and definitive detection of Salmonella in foods by PCR

The BAX^™ system for screening Salmonella is one of the first commercial PCR-based systems for the detection of food-borne pathogens. It is able to give a confirmed result within 28 h. There was 98·6% and 95·8% agreement between the BAX^™ system and conventional cultural analysis for the detection of Salmonella in artificially inoculated and uninoculated food samples, respectively. In both cases, the BAX^™ system generated more positive detections than cultural analysis. The speed of assay, ease of use and high specificity and sensitivity of the BAX^™ system for the detection of food-borne Salmonella make it an attractive method for routine food microbiology laboratories.