沙门菌、志贺菌和副溶血性弧菌的多重PCR快速检测技术的建立与应用

目的建立快速同时检测沙门菌、志贺菌和副溶血弧菌的多重聚合酶链反应(PCR)技术。方法根据沙门菌hilA基因、志贺菌ipaH基因及副溶血性弧菌tdh基因序列设计引物,进行PCR扩增及电泳检测。同时优化反应体系,测定特异性和灵敏度。结果该多重PCR技术能在8 h内同时检测3种目的菌,通过检测其他9种常见食源性致病菌,结果表明该方法特异性好,多重PCR检测灵敏度分别为:沙门菌104cfu/mL,志贺菌102cfu/mL,副溶血性弧菌104cfu/mL。并进行了人工模拟食品和粪便样品的检测。结论初步建立能在8h内快速、灵敏、特异地同时测定沙门菌、志贺菌和副溶血性弧菌的多重PCR检测技术。     

一起产气荚膜梭菌食物中毒事件的实验室检测分析

目的应用3种方法对一起食物中毒事件中的可疑样品进行检测和结果分析。方法应用实时荧光PCR、数字PCR和平板计数培养法检测食物中毒事件中的可疑食品盐水猪肝,并对分离菌株用脉冲场凝胶电泳（PFGE）进行分型。 应用细菌生化试验、实时荧光PCR鉴定可疑食品中的产气荚膜梭菌;应用数字PCR对毒力基因进行绝对定量。结果可疑食品产气荚膜梭菌平板计数值为4 400 000 CFU/g,并分离到22个产气荚膜梭菌单菌落,实时荧光PCR检测可疑食品样本产气荚膜梭菌plc和cpe基因阳性,其中3个菌落plc+/cpe+,19个菌落plc+/cpe–;数字PCR检测可疑食品样本plc基因绝对定量值为1064 拷贝/μl;22个单菌落分为2种PFGE带型。结论通过3种方法检测可疑食品中产气荚膜梭菌,说明该起食物中毒可能由产气荚膜梭菌导致。     

沙门菌、志贺菌和副溶血性弧菌的多重PCR快速检测技术的建立与应用

目的建立快速同时检测沙门菌、志贺菌和副溶血弧菌的多重聚合酶链反应（PCR）技术。方法根据沙门菌hilA基因、志贺菌ipaH基因及副溶血性弧菌tdh基因序列设计引物,进行PCR扩增及电泳检测。同时优化反应体系,测定特异性和灵敏度。结果该多重PCR技术能在8h内同时检测3种目的菌,通过检测其他9种常见食源性致病菌,结果表明该方法特异性好,多重PCR检测灵敏度分别为：沙门菌10^4cfu／mL,志贺菌10^2cfu／mL,副溶血性弧菌10^4cfu／mL。并进行了人工模拟食品和粪便样品的检测。结论初步建立能在8h内快速、灵敏、特异地同时测定沙门菌、志贺菌和副溶血性弧菌的多重PCR检测技术。     

Detection of Listeria monocytogenes from food samples by PCR after IMS-plating

For rapid, accurate and sensitive detection of Listeria monocytogenes in food samples, colonies developed on the selective agar (Oxford agar) after immunomagnetic separation (IMS) were subjected to polymerase chain reaction (PCR) assay with the prf A1-2 primer pair. The proposed assay system was shown experimentally to be capable of specifically detecting the bacteria from food samples contaminated at more than 10(2) cfu/g. However, the enrichment culture after a short period of 16 h with the appropriate selective broth was needed before IMS-plating, because the bacterial contents in most actual food were as low as less than 10(2) cfu/g. However, even if the enrichment cultivation was employed before IMS, L. monocytogenes was detected within 3 days.     

Sample preparation methods for PCR detection of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes on beef chuck shoulder using a single enrichment medium.

To improve the utility of the polymerase chain reaction (PCR) for food samples, methods for preparing template DNA were developed to remove PCR inhibitors. Beef chuck shoulder medallions, artificially contaminated, individually or in combination, with Escherichia coli serotype O157:H7 strain FSIS 45753-35, Salmonella typhimurium DT104 strain 13HP, or Listeria monocytogenes strain Scott A at concentrations of 10, 1 and 0.5 cfu/cm(2)were swabbed with a sponge, and the sponges were enriched for 18 h at 37 degrees C in universal pre-enrichment broth (UPB). Enriched broth cultures (EBC), cell pellets (CP), or phosphate-buffered saline-washed cell pellets (PBSCP) from enriched sponge samples were compared for detection of E. coli O157:H7, S. typhimurium DT104, or L. monocytogenes by the PCR using the BAX(TM)system. Recovery of the three organisms was effective for detection of each pathogen at initial levels of 10, 1 and 0.5 cfu/cm(2)when inoculated separately, or in combination, onto the beef samples. Use of EBC, CP, or PBSCP of sponge-swabbed samples eliminated problems associated with inhibition of the PCR by food components, time-consuming extraction of DNA, and inhibition due to large amounts of non-target DNA derived from the food. The procedure involving enrichment of sponge-swabbed beef samples in UPB followed by PCR amplification using EBC with the BAX system is the most efficient and simple method for detection of E. coli O157:H7, S. typhimurium DT104, and L. monocytogenes.     

Rapid, sensitive, and validated method for detection of Salmonella in food by an enrichment broth culture - nested PCR combination assay

A rapid nested PCR assay for detection of Salmonella from food was developed. The sensitivity of the assay developed was comparable to the traditional culture based methods with an advantage in reduction of assay time. The assay procedure with artificially contaminated samples was able to detect as low as 4CFU Salmonella/25g of food samples (sprout, carrot, cucumber and poultry meat). With two synthetic primers of 26 mer TS11 and 25 mer TS4, a 1.2kb fragment was amplified which served as a template for amplification of final 375bp product using TS11 and TS5 primers. No non-specific amplification from the native microbial flora of food samples was observed. The reaction generates a single band specific to Salmonella which allows the analyst to interpret data at ease and without any confusion. Enriched broth serves as template for the reaction which removes labour intensive DNA isolation procedures. In case of artificially contaminated samples, 6h enriched lactose broth can serve as template. However, for market samples where the organisms are under environmental stress, it is desirable to use template from Rappaport Vasiliadis medium. The assay also employes internal amplification control, which is amplified into a 300bp fragment and thus serves as positive control for the reaction and any possibility of false negative due to inhibitory action of food components on PCR reaction can be ruled out.     

16S rRNA通用引物在血小板制品细菌污染检测中的应用

目的评价16S rRNA通用引物在快速检出血小板制品中污染细菌的实用性,寻找新的能够快速、准确地检出血小板中污染细菌的方法。方法用分子生物学方法提取样本中细菌DNA,用设计合成的16S rRNA和16S—23S rRNA基因区间引物进行扩增,产物经HaeⅢ酶切,酶切片段与血小板污染常见菌的酶切图谱对比,确定有无污染及污染菌种类。结果16S rRNA通用引物法可以在4h内完成全部检测,其中在保存24h的血小板标本中未检出污染菌,保存48h的血小板标本中有2份分别检出了金黄色葡萄球菌和枯草芽孢杆菌,与全自动细菌培养仪的结果一致。结论16S rRNA通用引物是一种快速、准确检出血小板制品中污染细菌的方法,结果可靠,具有较高的临床应用价值。     

一起疑似产气荚膜梭菌食物中毒事件的实验室检测分析

目的 通过一起疑似产气荚膜梭菌食物中毒事件的检测分析,为类似食物中毒事件提供诊断依据。方法 采用多重实时荧光定量PCR方法和基因芯片检验方法快速筛查识别病原菌,根据初筛结果进行常规的细菌分离培养、生化鉴定和质谱分析,对分离到的菌株采用脉冲场凝胶电泳(PFGE)技术进行分子分型,采用BioNumerics 8.0软件对菌株的PFGE指纹图谱进行聚类分析。结果 多重实时荧光定量PCR方法提示4份粪便标本中均有产气荚膜梭菌和基因芯片检验方法提示1份鸡肝食品中有产气荚膜梭菌,并通过常规的细菌分离培养、生化鉴定和质谱分析从2份剩余鸡肝和4份腹泻患者粪便中分离到产气荚膜梭菌,其中只有3份粪便标本中产气荚膜梭菌计数>1.0×10⁶ CFU/g,但通过PFGE聚类分析从6份标本中检出的菌株图谱完全一致。结论 通过从食物和腹泻标本中检出PFGE图谱相似度100%的产气荚膜梭菌,说明此次食物中毒可能是由被产气荚膜梭菌污染的鸡肝导致的,今后要提高由厌氧菌引起的食物中毒的认识水平,防止漏检。     

模拟粪便标本单增李斯特菌和伊氏李斯特菌TaqMan双重实时荧光定量-聚合酶链反应检测方法

目的建立一种TaqMan双重实时荧光定量-聚合酶链反应(real-time PCR),用于模拟粪便标本中单增李斯特菌和伊氏李斯特菌的快速检测。方法以单增李斯特菌特异基因hly和伊氏李斯特菌特异基因smcL作为靶基因,合成2对引物和其相应的荧光探针,制作标准曲线,建立从模拟粪便标本中直接检测单增李斯特菌和伊氏李斯特菌的TaqMan双重real-time PCR。利用李斯特菌属中其他种李斯特菌及常见致病菌验证引物、探针的特异性;通过制备模拟粪便标本并对其进行二次增菌后,分别提取DNA,采用TaqMan双重real-time PCR进行检测,以达到快速检测单增李斯特菌和伊氏李斯特菌的目的。结果采用本研究建立的TaqMan双重real-time PCR对模拟粪便标本的检测结果显示特异性良好,与其他种李斯特菌和其他病原菌均无交叉反应。模拟粪便标本中单增李斯特菌和伊氏李斯特菌的检测下限分别为2.45×103cfu/g和2.92×103cfu/g;模拟粪便标本在3 h内可得出检测结果,当模拟粪便标本中单增李斯特菌含量为6 cfu/g和伊氏李斯特菌含量为5 cfu/g时,经过增菌后使用双重real-time PCR可检测出阳性结果。结论本研究建立了以单增李斯特菌的特异基因hly和伊氏李斯特菌的特异基因smcL为靶基因的TaqMan双重realtime PCR检测方法,该方法具有特异性好、敏感性高、快速易操作等优点,可用于临床、食品及环境标本的快速诊断,以及我国人群中单增李斯特菌和伊氏李斯特菌的携带或感染状况的调查分析。     

Rapid detection of Listeria monocytogenes by a PCR assay specific for an aminopeptidase

Specific and rapid detection of Listeria monocytogenes is very important with regard to food safety since all other species of Listeria appear to be non-pathogenic to humans. Conventional microbiological detection methods are very time consuming. The polymerase chain reaction (PCR) is one of the most promising techniques for rapid detection of micro-organisms in food products. We have developed a PCR assay, specific for L. monocytogenes, based on the gene encoding an aminopeptidase, which previously has not been described for this species. The L. monocytogenes aminopeptidase shares strong sequence similarity with aminopeptidase C from Streptococcus thermophilous, Lactobacillus lactis, Lactobacillus helveticus, and with a cysteine proteinase from Saccharomyces cerevisiae. Polymerase chain reaction primers were synthesized based on the DNA sequence of the aminopeptidase gene. A 90 bp product was apparent with all L. monocytogenes strains tested but not with other species of Listeria or other bacterial genera. The PCR assay, which is performed directly from whole bacterial cells, does not involve DNA purification and can be conducted in 4 h. It provided positive identification of L. monocytogenes in mixed culture.     

应用聚合酶链反应法检测食品中的沙门菌

目的建立食品中沙门菌聚合酶链反应（PCR）的快速检测方法。方法根据沙门菌invA基因序列设计引物;氯化镁孔雀绿选择性增菌0到8h后,煮沸法提取DNA,用PCR扩增电泳。结果PCR法能特异性检测出食品中的沙门菌,扩增片段为389bp,增菌前的检出限为10^2CFU／g,增菌后为2CFU／25g。结论应用PCR检测沙门菌具有快速、特异、灵敏和简便的特点。     

Two methods for construction of internal amplification controls for the detection of Escherichia coli O157 by polymerase chain reaction

For the detection of food born bacteria by polymerase chain reaction (PCR) in food products, an internal amplification control (IAC) is required in order to prevent false negative results that might be caused by PCR inhibitors. In the present study, two IACs were constructed using two different methods. These IACs were designed in a way that the same primer pair can be used to amplify the target DNA and coamplify the IAC. The first IAC with a size of approximately 200 bp was constructed by deleting a part of the amplicon of the original target DNA (500 bp) between the two primer sites to produce an IAC smaller than the target DNA. The second IAC with a size of approximately 600 bp was synthesized in a one step PCR reaction. The primers used in this reaction possessed 5' over-hanging ends, which were identical to the primers used in the diagnostic reaction, whereas their 3' ends were complementary to the (pUC19) predetermined DNA sequence of defined length and sequence. The concentration of IACs appeared to be critical. Too much IAC DNA template would out-compete the target DNA template, thus giving a false negative result. However the use of an optimal IAC concentration increased the reliability of the PCR assays and appeared to be useful for food diagnostics.     

巢式聚合酶链式反应检测布鲁氏菌核酸DNA方法的建立

目的建立一种具有灵敏性高,特异性强的巢式聚合酶链式反应(PCR)方法检测血液标本中布鲁氏菌核酸DNA。方法使用细菌基因组提取试剂盒提取纯菌核酸DNA;使用血液等组织基因组核酸DNA提取试剂盒提取血液标本核酸DNA,对提取的核酸DNA先行常规PCR预扩增,以扩增的PCR产物为模板进行荧光定量PCR第二次扩增(即巢式PCR)。对纯菌提取的核酸DNA进行灵敏度和和特异性测试,构建巢式PCR的Ct值与核酸DNA拷贝数之关系曲线;检测临床血液标本核酸DNA,同时比较常规两种PCR方法检测结果。结果常规PCR检测的灵敏度为512个核酸DNA拷贝数;巢氏PCR检测有效范围为921.6 ng/μl^6.8 fg/μl,对应的Ct值为12.04~37.50,其指数关系为:y=(e-0.695x)×1012;R2=0.9986,巢式PCR扩增效率为2.28×109倍,检测限为2个布鲁氏核酸DNA拷贝数。巢式PCR的灵敏度为91.67%,特异度为93.10%,阳性预测值为91.67%,阴性预测值为93.10%。对一起羊养殖场采集的25份血液标本应用巢式PCR方法检测,结果阳性率为92.00%(23/25);27份健康人群血液标本没有检测出(无Ct值)。结论巢式PCR具有较好的灵敏性和特异性,特别适合于血液标本布鲁氏菌核酸DNA的检测。     

Detection of bacteria in platelet concentrates: comparison of broad-range real-time 16S rDNA polymerase chain reaction and automated culturing

BACKGROUND: Based on real-time polymerase chain reaction (PCR) technology, a broad-range 16S rDNA assay was validated and its performance was compared to that of an automated culture system to determine its usefulness for rapid routine screening of platelet concentrates (PCs). STUDY DESIGN AND METHODS: The presence of bacteria in pooled PCs was routinely assessed in an automated culturing system (BacT/ALERT, bioMerieux). The PCR assay was performed with DNA extracted from the same samples as used for culturing. DNA extraction was performed with a automated extraction system (MagNA Pure, Roche Diagnostics). PCR amplification was performed with a set of universal primers and probe targeting eubacterial 16S rDNA. RESULTS: A total of 2146 PCs were tested. Eighteen (0.83%) samples were found to be contaminated. These samples were positive for the presence of bacteria by both methods. All contaminants were identified as bacteria belonging to the common human skin flora. These included Propionibacterium spp. (n = 7), Staphylococcus spp. (n = 6), Bacillus spp. (n = 2), Micrococcus spp. (n = 2), and Peptostreptococcus spp. (n = 1). Estimation of the bacterial load in PCs by real-time PCR showed that the initial levels of contamination varied between 13.6 and 9 x 10(4) colony-forming unit equivalents per PCR procedure. CONCLUSIONS: Compared to culture in the BacT/ALERT system, the PCR assay had a sensitivity of 100 percent and a specificity of 100 percent. This real-time PCR assay has a much shorter turnaround time of 4 hours, which offers the possibility to test and obtain results on PCs before release or the day they are transfused. This would permit the withdrawal of contaminated PCs before transfusion.     

Evaluation of an extended diagnostic PCR assay for detection and verification of the common causes of bacterial meningitis in CSF and other biological samples

A seminested polymerase chain reaction (PCR)-based diagnostic assay was evaluated for detection and verification of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Steptococcus agalactiae and Listeria monocytogenes in cerebrospinal fluid (CSF) and other biological samples. A general bacterial amplicon from the 16S rRNA gene was amplified in a first step, and species-specific regions in a second. The detection level was 4 fg DNA/reaction, corresponding to about one bacterial genome per reaction tube. Sample preparations (Dynabeads DNA DIRECT kit) were assayed from 140 bacterial strains suspended in saline. In CSF the detection level for bacteria was 10(3)CFU ml-1for N. meningitidis, H. influenzae and S. pneumoniae, 10(4)CFU ml-1for Escherichia coli and 10(5)CFU ml-1for S. agalactiae and L. monocytogenes. The detection levels for these bacteria were the same in the other tested biological samples, like blood with or without culture media. Clinical CSF samples were evaluated from 71 patients with proven bacterial meningitis, as were 61 CSF samples from individuals without bacterial meningitis. The diagnostic sensitivity of the assay in detecting bacteria in general was 0.97, and for the specific species in the clinical CSF samples 0.87-0.94. The specificity was 1.0 for detecting bacteria in general. Some cross-reactions were noted within the streptococcus group. The PCR results were verified by banding patterns of Hae III digested PCR products.     

分子水平快速诊断败血症致病菌的研究

目的通过合成所有细菌共有的16SrRNA基因高度保守区引物,进行PCR扩增,对8种标准菌株、10种40株临床分离细菌进行了研究,探索快速而准确诊断败血症的新方法,以达到早期治疗的目的。方法通过PCR技术,对40例败血症患者进行了分析,完成了对败血症患者的早期诊断。结果8种标准菌株、10种40株临床分离细菌均获得308bp扩增产物。结论PCR检测方法是一种易于推广的败血症早期诊断方法。     

6种食品致病菌的多重PCR检测

目的寻求食物中毒诊断及食品检测的有效方法。方法建立多重PCR体系一次检测多种食品致病菌方法。结果可以一次检测出肠毒性大肠埃希菌(E.coli-ETEC),蜡样芽胞杆菌(Bacilluscereus),沙门菌属(Salmonellaspp),大肠埃希菌O157:H7(E.coli-O157:H7),志贺菌属(Shigellaspp),霍乱弧菌(Vibriocholerae)等菌属或菌。结论多重PCR体系检测6种菌DNA的灵敏度最低为10.30fg,与单独的PCR检测的灵敏度相同。将它做成体外基因诊断试剂盒,将成为细菌性食物中毒诊断及食品检测的有效工具。     

Rapid identification of Pseudomonas aeruginosa from ocular isolates by PCR using exotoxin A-specific primers

The purpose of this research was to evaluate the use of PCR for the identification of ocular isolates of Pseudomonas aeruginosa by using primers specific to the exotoxin A gene of the bacteria. Genomic DNA was obtained from ocular microbial isolates of keratitis patients. Primers were designed based on the published sequence of the exotoxin A gene of P. aeruginosa. Using the primers designed, PCR reactions were performed on the DNA samples. The PCR was also examined for its specificity and sensitivity. In addition, a direct PCR using heating method was attempted on P. aeruginosa with no separate DNA extraction step. ATCC strains of P. aeruginosa were included as positive controls. The rest of the bacteria other than P. aeruginosa served as negative controls. A single band was obtained when analysed on agarose gel electrophoresis only from samples that contained genomic DNA of P. aeruginosa. The direct PCR method was also successful with the same band produced from the amplification. The whole process was completed within 4 h. The direct PCR amplification targeting at the exotoxin A gene of P. aeruginosa is potentially a rapid, specific, sensitive and relatively simple method for the identification of ocular isolates of P. aeruginosa.     

一起甲型副伤寒沙门菌污染水源所致腹泻暴发疫情的病原学调查

目的对一起腹泻暴发疫情进行病原学调查,以便明确病因、进行有效的预防控制。方法采用荧光PCR快速检测、常规病原学分离培养、噬菌体裂解试验、ATB微生物自动鉴定系统等方法对疑似患者、环境水源水和饮用水采样,进行病原学的检测和鉴定。结果经荧光PCR检测与病原分离培养鉴定,来自患者粪便、血液、水源水和饮用水共12份疑似标本中,10份为甲型副伤寒沙门菌阳性,检出率为83.33%,其中4份来自粪便标本,4份来自血,1份来自水源水,1份来自饮用水。10株不同来源甲型副伤寒沙门菌的血清学分型、生化反应、药物敏感性、噬菌体裂解试验均相同。结论这是一起由甲型副伤寒沙门菌水源污染所致的腹泻暴发疫情。该菌的检出为本地区腹泻病原谱增加了一种新的病原菌。     

16S rDNA基因芯片检测临床常见感染性细菌

目的 提高细菌和临床微生物检测的速度和准确性，建立含有20种细菌探针在内的感染性细菌检测用基因芯片模型。方法 使用16S rDNA克隆探针和合成的寡核苷酸探针两种，利用点样仪制成基因芯片。细菌DNA经过16S rDNA通用引物扩增后与芯片上的探针杂交，然后用荧光扫描仪检测信号。结果 基因芯片能够用于细菌检测，克服探针具有广泛和灵敏的特点，但是交叉反应明显；寡核苷酸探针具有较高的特异性，但是灵敏度稍差。结论 cDNA探针和寡核苷酸探针结合或设计几个不同的探针来指向同一株细菌很可能是将来基因芯片的检测方向。