Identification of enteric pathogens by heat shock protein 60 kDa (HSP60) gene sequences

A highly specific and reproducible approach for the simultaneous detection of enteric pathogenic bacteria was developed using bacterial hsp60 gene and molecular biological tools. A single pair of universal primers was derived from the highly conserved sequence of hsp60 genes encompassing a 600-bp hypervariable region. PCR amplification followed by either dot blot hybridization or restriction enzyme digestion performed on 38 enteric bacteria indicated that this approach could differentiate not only different genera such as Campylobacter, Yersinia and Vibrio, but also species that are closely related genetically, such as between C. jejuni and C. coli, or between Salmonella and Shigella or Escherichia coli.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes.

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

Development of oligonucleotide primers for the specific PCR-based detection of the most frequent Enterobacteriaceae species DNA using wec gene templates

Oligonucleotide primers were designed for the PCR-based detection of the wec gene cluster involved in the biosynthetic pathway leading to the production of enterobacterial common antigen (ECA). Escherichia coli DNA was detected using wec A, wec E, and wec F gene primers. The wec A primers were specific for E. coli. The wec E and wec F primers enabled the detection of the most frequent species of the Enterobacteriaceae found in blood and urine specimens as well as in water. The sensitivity of the assay was approximately 1.2 x 102 bacteria/mL of water. Thus, these primers represent an important step in the molecular diagnosis of major Enterobacteriaceae infections. Their role in the routine testing of contamination in drinking water and food may prove to be very useful. The DNA of Enterobacteriaceae species is detected in a first step PCR, followed by specific identification of important pathogens like E. coli O157, Shigella spp., Salmonella spp., and Yersinia spp.     

基因芯片技术检测3种食源性致病微生物方法的建立

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 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。     

致病性大肠埃希菌和沙门菌毒力岛基因的双重PCR检测方法的建立

目的实现对致病性大肠埃希菌（E．coli）、沙门菌（Salmonella）的同时检测,建立快速灵敏的双重PCR检测方法。方法以致病性大肠埃希菌和沙门菌毒力岛基因为研究对象,根据GenBank发表的大肠埃希菌和沙门菌毒力岛基因序列,分别设计合成了大肠埃希菌毒力岛irpl、irl）2和fyuA,沙门菌毒力岛mgtC、sseL和sopB等6对引物,以禽致病性大肠埃希菌（CVCC1565）菌株和沙门菌（ATCC9150）菌株的核酸混合物为模板,经引物特异性试验,引物组合,成功建立了快速鉴别检测致病性大肠埃希菌和沙门菌的双重PCR方法。结果特异性试验结果显示,引物irpl、irp2和fyuA仅能扩增出大肠埃希菌（CVCC1565）的特异性片段,大小分别是799、414和948bp;引物mgtC、sseL和sopB仅能扩增出沙门菌（ATCC9150）的特异性片段,大小分别是500、269和1000bp。敏感性试验结果表明大肠埃希菌和沙门菌的最低检测限分别为2．2×101CFU／mL和2．0×101CFU／mL。结论本研究建立的双重PCR方法具有特异性强、敏感性高、快速简便等特点,可用于致病性大肠埃希菌和沙门菌的联合检测与鉴别诊断。     

Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in mutiplex format

AIMS: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. METHODS AND RESULTS: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC-d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which co-amplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 10(4) CFU ml(-1) (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre-enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. CONCLUSIONS: The developed mPCR assay provides specific detection of S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.     

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.     

Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. In this paper, we described the development of a real-time PCR assay to detect the presence of Escherichia coli O157:H7 using these fluorogenic reporter molecules. MBs were designed to recognize a 26-bp region of the rfbE gene, coding for an enzyme necessary for O-antigen biosynthesis. The specificity of the MB-based PCR assay was evaluated using various enterohemorrhagic (EHEC) and Shiga-like toxin-producing (STEC) E. coli strains as well as bacteria species that cross-react with the O157 antisera. All E. coli serotype O157 tested was positively identified while all other species, including the closely related O55 were not detected by the assay. Positive detection of E. coli O157:H7 was demonstrated when >10(2) CFU/ml was present in the samples. The capability of the assay to detect E. coli O157:H7 in raw milk and apple juice was demonstrated. As few as 1 CFU/ml was detected after 6 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli O157:H7 in food and environmental samples.     

Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella

Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We developed a real-time PCR assay to detect the presence of Salmonella species using these fluorogenic reporter molecules. A 122-base-pair section of the himA was used as the amplification target. Molecular beacons were designed to recognize a 16-base-pair region on the amplicon. As few as 2 colony-forming unit (CFU) per PCR reaction could be detected. We also demonstrated the ability of the molecular beacons to discriminate between amplicons obtained from similar species such as Escherichia coli and Citrobacter freundii in real-time PCR assays. These assays could be carried out entirely in sealed PCR tubes, enabling fast and direct detection of Salmonella in a semiautomated format.     

Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene

AIMS: A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. METHODS AND RESULTS: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method. CONCLUSIONS: The developed real-time PCR is suitable for rapid quantification of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.     

基于免疫磁分离的三重荧光定量PCR检测食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌

【目的】开发一种同时对食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌快速、灵敏、准确的检测方法。【方法】利用特异性免疫磁球,在37°C条件下从250 m L猪肉增菌液体系中边富集边循环捕获目标菌。快速提取DNA后,利用特异性的引物与探针,对3种食源性致病菌进行三重荧光定量PCR检测。【结果】针对沙门氏菌、志贺氏菌和金黄色葡萄球菌的检测限分别达到2.0、6.8和9.6 CFU/g。方法总体灵敏度、特异性和准确度达到99.2%、100%及99.5%。对151份实际样品进行检测,与国标(GB/T 4789.4-2010、GB 4789.5-2012和GB/T4789.10-2010)方法的检测结果相比,金黄色葡萄球菌有一例阴性偏差。【结论】开发的基于免疫磁分离的三重荧光定量PCR方法,能够在8 h内完成对食品中3种致病菌检测,并且灵敏度高、特异性好、检测准确,可以作为快速应对此类食品安全突发事件的检测手段。     

餐饮及相关食品中病原菌活菌多重实时荧光PCR检测方法的研究与开发

为了应对餐饮等食品中病原菌快速检测的需求、研究建立病原菌筛查方法,选取痢疾志贺氏菌（Shigella dysenteriae）、金黄色葡萄球菌（Staphylococcus aureus）、副溶血性弧菌（Vibrio parahaemolyticus）、阴沟肠杆菌（Enterobacter cloacae）、产气肠杆菌（Enterobacter aerogenes）、沙门氏菌（Salmonella）、蜡样芽胞杆菌（Bacillus cereus）、大肠埃希氏菌O157∶H7（Escherichia coli O157∶H7）、单核细胞增生李斯特氏菌（Listeria monocytogenes）等9种病原菌开展多重实时荧光PCR方法研究工作。为了节约预增菌时间与提升检测效率,研发了适用于多种病原菌预增菌的通用型培养基,采取高温裂解法提取菌液核酸,利用PMA染料灭活死亡细菌DNA,筛选出活菌DNA,采用多重实时荧光PCR技术检测目标菌,该方法可在16 h内完成检测,对于目标病原菌的检测低限可达103 CFU·mL-1。     

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods wasdeveloped. The protocol used a universal culture medium and the same PCR conditions with 13sets of specific primers. The 13 species of foodborne pathogens examined were Escherichiacoli, E. coli- ETEC, E. coli -O157:H7, Shigella spp. , Salmonella spp. , Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V.parahaemolyticus, V. vulnificus , Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus . No interference was observed using the PCR assay when foodsample was artificially inoculated with each individual bacterial species. Twelve different seafoodsamples and two soft cheese samples without artificial inoculation were examined by thisprotocol. Vibrio vulnificus, Salmonella spp. , E. coli,Listeria monocytogenes and Bacillus cereus were detected in some foods.Internal probe hybridization and nested PCR procedures were used to confirm the above findings.     

Glutamate decarboxylase genes as a prescreening marker for detection of pathogenic Escherichia coli groups.

The enzyme glutamate decarboxylase (GAD) is prevalent in Escherichia coli but few strains in the various pathogenic E. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA and gadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagic E. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using a gadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR to gadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx(1) and stx(2) was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic E. coli groups.     

多种食源性致病菌检测的多重PCR 方法的研究

目的:利用多重PCR技术,建立可以同时检测多种食源性致病菌的多重PCR方法。方法:分别选择沙门氏菌invA基因,志贺氏菌的ipaH基因,单核细胞增生李斯特氏菌的hlyA基因,大肠杆菌O157:H7的eaeA基因,副溶血弧菌的toxR基因,设计多重PCR引物,建立多重PCR检测体系,并对该体系进行特异性和灵敏度实验。结果:通过对19株菌株进行实验,所有的目标菌株均为阳性,而其余菌株为阴性。对多重PCR体系的灵敏度进行考察,沙门菌的灵敏度为5000 CFU/mL;志贺氏菌的灵敏度为5500CFU/mL;单核细胞增生李斯特氏菌的灵敏度为5200 CFU/mL;O157:H7的灵敏度为5000CFU/mL;副溶血弧菌的灵敏度为6300CFU/mL。结论:建立的多重PCR体系能实现多种致病菌同时检测。     

环介导等温扩增联合横向流动试纸条可视化检测志贺氏菌

【目的】将环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)联合应用,建立一种可应用于志贺氏菌快速检测的LAMP-LFD技术。【方法】以福氏志贺氏菌的侵袭性质粒抗原H(ipa H)基因为检测靶标设计3对特异性引物(其中上游内引物Sfl-ipa H-FIP由生物素标记),进行LAMP反应;同时设计1条异硫氰酸荧光素(FITC)标记的探针Sfl-ipa H-HP,与获得的LAMP产物进行特异性杂交,杂交产物经LFD完成检测。【结果】优化后的LAMP反应条件为63°C 40 min,加上LFD结果判读共需50 min。LAMP-LFD方法能够特异性检测出福氏志贺氏菌,而对肠炎沙门氏菌等其它4种导致腹泻的致病菌和创伤弧菌等5种常见食物源性致病菌,以及4株不同大肠杆菌的检测结果呈阴性。该方法针对福氏志贺氏菌的检测灵敏度为1.0×10~2 CFU/m L或4 CFU/反应,针对人工污染鲤鱼肠组织的检测灵敏度是5.0×10~2 CFU/m L,是以LAMP外引物Sfl-ipa H-F3/Sfl-ipa H-B3的常规PCR方法的100倍。【结论】建立的LAMP-LFD技术具有操作简单、检测快速准确、检测成本低等优点,有望在志贺氏菌的常规监测和即时检测中被普及使用。     

PCR amplification of the fimA gene sequence of Salmonella typhimurium, a specific method for detection of Salmonella spp.

The goal of this study was to evaluate the suitability of the fimA gene amplification by PCR as a specific method for detection of Salmonella strains. Salmonella typhimurium and other pathogenic members of the family Enterobacteriaceae produce morphologically and antigenically related, thin, aggregative, type 1 fimbriae. A single gene, fimA, encodes the major fimbrial unit. In order to obtain higher specificity, we have selected a series of primers internal to the fimA gene sequence and have developed a PCR method for detecting Salmonella strains. A collection of 376 strains of Salmonella comprising over 80 serovars, isolated from animals and humans in Canada, have been used to evaluate this PCR method. Forty non-Salmonella strains were also tested by the same procedure. Cultures were screened by inoculating a single colony of bacteria directly into a PCR mixture containing a pair of primers specific for the fimA gene. The specific PCR product is an 85-bp fragment which was visualized by polyacrylamide gel electrophoresis and ethidium bromide staining. All Salmonella strains gave positive results by the PCR. Feed and milk samples contaminated by Salmonella strains were also detected by this procedure. The detection of all Salmonella strains tested and the failure to amplify the fragment from non-Salmonella strains confirm that the fimA gene contains sequences unique to Salmonella strains and demonstrate that this gene is a suitable PCR target for detection of Salmonella strains in food samples.     

PCR for Detection of Shigella spp. in Mayonnaise

The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasive Escherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.     

Use of two 16S DNA targeted oligonucleotides as PCR primers for the specific detection of Salmonella in foods

A 16S DNA targeted polymerase chain reaction (PCR) method specific for the detection of Salmonella isolates with various serotypes was developed. The primers used for such a PCR method were 16SF1 and 16SIII. 16SF1 is the reverse and complementary strand of 16SI which has been shown to be able to hybridize with Salmonella and Citrobacter spp. 16III on the other hand, is able to hybridize with Klebsiella and Serratia spp. in addition to Salmonella. Although 16SF1 and 16SIII were not specific to Salmonella only, when they were used as PCR primers, only the Salmonella isolates could be specifically detected. The interference from Citrobacter, Klebsiella and Serratia spp. could be prevented. None of the other non- Salmonella isolates including strains of the family of Enterobacteriaceae closely related to Salmonella would generate the false-positive reaction. When this PCR system was used for the detection of Salmonella cells artificially contaminated in food samples, results obtained were satisfactory. A detection limit of N × 10⁰ cells per assay could be obtained.