大肠杆菌肠毒素基因多重PCR检测方法的建立

目的 建立一种快速检测大肠杆菌耐热肠毒素(heat-stable enterotoxin, STa, STb)和不耐热肠毒素(heat-labile enterotoxin, LT-Ⅰ, LT-Ⅱ)基因的多重PCR方法。方法 参照文献合成四对可扩增产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli, ETEC)耐热肠毒素基因(estA、estB)和不耐热肠毒素基因(elt-Ⅰ、elt-Ⅱ)的特异性引物,通过反应条件的优化,敏感性、特异性试验和临床样品检测,建立检测大肠杆菌肠毒素的多重PCR方法。结果 用所建立的多重PCR方法可特异性扩增出estA(229 bp)、estB(480 bp)、elt-Ⅰ(605 bp)和elt-Ⅱ(300 bp)基因片段,最低检出量分别为2.55×10¹ CFU/μL、2×10¹ CFU/μL、2×10¹ CFU/μL和2.47×10³ CFU/μL。从22株大肠杆菌分离株中检测到estA基因(2/22),elt-Ⅱ基因(3/22),未检测到estB和elt-Ⅰ基因,检测结果与常规PCR检测结果一致。结论 建立了检测大肠杆菌肠毒素基因(estA、estB、elt-Ⅰ和elt-Ⅱ)的多重PCR方法,该方法具有良好的特异性和敏感性,能够满足对细菌培养物的检测要求。     

一种直接检测结核病痰标本的PCR技术的建立与评价

目的建立并评价聚合酶链式反应(polymerase chain reaction,PCR)在结核病痰标本检测中的应用价值。方法根据结核分枝杆菌复合体IS6110序列设计引物INS1和INS2,并建立PCR反应体系和反应条件。运用PCR方法分别检测标准菌株、结核分枝杆菌PCR检测标准品和拟诊结核病患者痰标本,采用痰涂片和细菌培养为对照。结果比较的统计学分析采用卡方检验。结果PCR方法对结核分枝杆菌、牛分枝杆菌、卡介苗标准株的最小检出浓度分别达到102,103,103个细菌/毫升,能够特异地检出结核分枝杆菌复合体。在PCR检测的574例拟诊病例中,PCR检测阳性病例241例,42%;痰涂片和细菌培养的阳性率分别为19.69%和26.31%,PCR检测阳性率高于传统细菌学检验方法,经χ2检验,差异具有显著统计学意义(χ2=103.67,P<0.01)。以痰培养结果为标准,计算PCR检测方法的敏感度为67.53%。结论PCR检测方法与传统的细菌学检测方法相比可以提高阳性标本检出率,而且具有快速、特异、简便的特点,有望成为结核病大规模筛查和临床快速检测方法。     

粪便中日本血吸虫虫卵DNA的提取及PCR检测方法的优化

目的建立一套系统、完整的粪便日本血吸虫虫卵DNA提取法及PCR优化体系。方法利用蛋白酶K和苯酚法从感染日本血吸虫尾蚴的家兔粪便中提取DNA,根据日本血吸虫基因（AF00369）设计特异性引物,以粪便中提取的DNA为模板,采用PCR方法扩增目的基因片段,对PCR方法的各种反应条件进行优化并采用有限稀释法推测PCR检测法可检测到的最低虫卵数。结果以感染家兔的粪便中提取的DNA为模板．用PCR方法可扩增到分子量大小约为400bp的特异性条带,测序结果经BLAST工具进行分析．与日本血吸虫基因（AF00369）比对的同源性为96％;25μl PCR反应体系的最优化反应条件：MgCl2 1．5μl;dNTP0．5μl;引物1．0μl;DNA模板5．0μl;TaqDNA聚合酶0．5μl。扩增程序为：94℃预变性5min、94℃变性30s、53℃退火30s、72℃延伸30s,30个循环、72℃延伸7min、4℃保存。PCR检测法可检测到的最低虫卵数为0．015个。结论成功建立感染家兔粪便中虫卵DNA的提取方法及PCR血吸虫基因片段检测方法,同时对PCR反应条件进行优化,为从分子角度检测日本血吸虫虫卵DNA提供科学的实验依据。     

应用PCR检测脑脊液中结核杆菌的研究

采用Manjunath等设计的一对引物,对人型结核杆菌H_(37)R_v以检测其敏感性为1pg,17种分支杆菌DNA仅人型、牛型及BCG出现240bp扩增带,其它均未出现扩增带.50例结脑csf作PCR检测结核菌与培养、涂片对比,阳性率分别为54%,6%和.8例非结核性脑膜炎均为阴性.结果表明,该引物具有较高的敏感性和特异性.应用PCR方法可以为结核性脑膜炎提供快速、敏感、特异的病原学诊断.     

ETEC肠毒素基因多重PCR检测方法的建立

肠毒素性大肠杆菌的致病性与其具有粘附性的菌毛和产肠毒素的能力密切相关。由于菌毛的血清型多而复杂 ,肠毒素只有不耐热肠毒素 (LT)和耐热肠毒素 (ST)两种 ,因此成为研究的对象。用三对扩增产物分别为 1 1 0bp、2 37bp、368bp的引物建立了检测LT和STⅠ、STⅡ毒素基因的多重PCR方法。扩增产物分别用HindⅢ、HincⅡ、Sau3AⅠ限制性内切酶酶切 ,均得与预期一致的 2个片段。对各个参考株的检测结果为 1 0 0 %符合。结果表明该多重PCR方法具有很好的特异性和敏感性。该方法可用于肠毒素性大肠杆菌腹泻病的辅助诊断以及大肠杆菌的分类检测     

PCR快速检测临床标本中结核杆菌的研究

本文用PCR(聚合酶链反应)技术检测238份临床标本中结核杆菌,并与培养及ELISA法进行对比观察。结果:用PCR扩增出123bp区带者142份(59.7％);培养出结核杆菌者55份(23.2％)。139份体腔液及尿标本还同时检测了抗PPD抗体,其中50份标本阳性(36％)。PCR检出率明显高于培养及ELISA(P<0.005)方法简便,快速,是一种检测结核杆菌的实用技术。     

猪伪狂犬病病毒与猪圆环病毒2型双重PCR检测方法的建立及初步应用

目的 为建立一种能同时鉴别猪伪狂犬病病毒(PRV)和猪圆环病毒2型(PCV2)混合感染的诊断方法。方法与结果 根据GenBank中公布的猪伪狂犬病病毒和圆环病毒2型的基因序列,分别设计一对特异性引物,扩增长度分别为612 bp和238 bp。将PCR产物进行测序,与PRV“Yangsan”株和PCV2“JX0301”株的同源性分别为98.6%和99.2%。通过反应条件的优化,建立同时检测PRV和PCV2的双重PCR方法。利用该方法对临床采集的78份疑似病料进行检测,其中59份为PCV2阳性,17份PRV阳性,其中11份为PRV和PCV2共感染。结论 建立的双重PCR检测方法可以用于PRV和PCV2的临床快速鉴别诊断和流行病学调查。     

粪便PCR检测法用于诊断日本血吸虫病应用价值的研究

目的 探讨PcR法检测日本血吸虫病的特异性、敏感性及与其他肠道寄生虫病的交叉反应情况.方法 采用PCR法对经病原学检查确诊的阳性粪样、非血吸虫病流行区的正常人粪样以及钩虫、蛔虫、鞭虫等肠道寄生虫感染粪样分别进行检测,评价PCR法的各项检测指标.结果 PCR法检测日本血吸虫病的敏感性和特异性均为100%,与钩虫、鞭虫、蛔虫等肠道常见寄生虫均无交叉反应.结论 粪便PCR法检测日本血吸虫病具有敏感性高、特异性强、交叉反应少等优点.具有较高的现场查病和临床诊断血吸虫病的应用价值.     

猪伪狂犬病病毒与猪圆环病毒2型双重PCR检测方法的建立及初步应用北大核心CSCD

目的为建立一种能同时鉴别猪伪狂犬病病毒(PRV)和猪圆环病毒2型(PCV2)混合感染的诊断方法。方法与结果根据GenBank中公布的猪伪狂犬病病毒和圆环病毒2型的基因序列,分别设计一对特异性引物,扩增长度分别为612bp和238bp。将PCR产物进行测序,与PRV"Yangsan"株和PCV2"JX0301"株的同源性分别为98.6%和99.2%。通过反应条件的优化,建立同时检测PRV和PCV2的双重PCR方法。利用该方法对临床采集的78份疑似病料进行检测,其中59份为PCV2阳性,17份PRV阳性,其中11份为PRV和PCV2共感染。结论建立的双重PCR检测方法可以用于PRV和PCV2的临床快速鉴别诊断和流行病学调查。     

聚合酶链反应直接检测粪便中志贺氏菌和侵袭性大肠杆菌

为探讨聚合酶链反应（ＰＣＲ）在腹泻病快速诊断的实用性，应用ＰＣＲ和长臂光敏生物素标记福氏２ａ的特异ＤＮＡ片段作探针，Ｓｏｕｔｈｅｒｎ印迹杂交法，检测志贺氏菌和侵袭性大肠杆菌（ＥＩＥＣ）共有的侵袭相关性基因（ｉａｌ），９株志贺氏菌和２株ＥＩＥＣ均存在特异的３２０ｂｐＤＮＡ片段。最小检测菌量为１００菌落形成单位（ｃｆｕ）。１９株非志贺氏菌均阴性。检测５８份粪标本，ＰＣＲ２３份阳性（３９．６％），粪培养１８份阳性（３１％）。全部实验，包括ＤＮＡ粗提取，仅需６～７小时。结果表明其敏感性高于粪培养，ＰＣＲ操作简便、快速、敏感、结果可靠，适用于腹泻病的快速诊断。     

Development of multiplex PCR for the detection of total coliform bacteria for Escherichia coli and Clostridium perfringens in drinking water

Multiplex PCR amplification of lacZ, uidA and plc genes was developed for the simultaneous detection of total coliform bacteria for Escherichia coli and Clostridium perfringens, in drinking water. Detection by agarose gel electrophoresis yielded a band of 876 bp for the lacZ gene of all coliform bacteria; a band of 147 bp for the uidA gene and a band of 876 bp for the lacZ gene of all strains of E. coli; a band of 280 bp for the p/c gene for all strains of C. perfringens; and a negative result for all three genes when tested with other bacteria. The detection limit was 100 pg for E. coli and C. perfringens, and 1 ng for coliform bacteria when measured with purified DNA. This assay was applied to the detection of these bacteria in spiked water samples. Spiked water samples with 0-1,000 CFU/ml of coliform bacteria and/or E. coli and/or C. perfringens were detected by this multiplex PCR after a pre-enrichment step to increase the sensitivity and to ensure that the detection was based on the presence of cultivable bacteria. The result of bacterial detection from the multiplex PCR was comparable with that of a standard plate count on selective medium (p=0.62). When using standard plate counts as a gold standard, the sensitivity for this test was 99.1% (95% CI 95.33, 99.98) and the specificity was 90.9 % (95% CI 75.67, 98.08). Multiplex PCR amplification with a pre-enrichment step was shown to be an effective, sensitive and rapid method for the simultaneous detection of these three microbiological parameters in drinking water.     

多重聚合酶链反应检测产毒性大肠杆菌三种毒力基因

目的提高普通的单基因聚合酶链反应（ＰＣＲ）检测产毒性大肠杆菌（ＥＴＥＣ）的时效。方法以ＥＴＥＣ４４８１３（ＳＴｐ＋）、ＥＴＥＣ１９４４９（ＳＴｈ＋）和ＥＴＥＣ４４８１５（ＬＴ＋）三个标准株为模板,建立了检测产毒性大肠杆菌多重ＰＣＲ扩增系统。结果杂交工程株Ｈ１０９０７（ＳＴｐ＋）,ＰＳＬＭ００４（ＳＴｈ＋）和ＰＭＭ０３０（ＬＴ＋）,杂交的结果都是阳性,５４株菌株经ＰＣＲ检测,其中ＬＴ＋８株、ＳＴｐ＋２株、ＳＴｈ＋７株、ＬＴ＋＋ＳＴｐ＋１０株、ＬＴ＋＋ＳＴｈ＋１０株、ＳＴｐ＋＋ＳＴｈ＋４株和ＬＴ＋＋ＳＴｐ＋＋ＳＴｈ＋１３株。结论用一次ＰＣＲ扩增可检测和鉴别ＥＴＥＣ,比单基因ＰＣＲ更加快速、经济。     

Comparison of multiplex PCR with serogrouping and PCR-RFLP of fliC gene for the detection of enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) comprise one of the six categories of diarrhoeagenic E. coli (DEC). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC). The identification of DEC cannot be based only on cultural and biochemical criteria, since they are indistinguishable from the non-pathogenic E. coli commonly found in human feces. Several PCR methods, with both single and multiple target genes, have been reported for detecting the different DEC pathotypes. In the present study five hundred E. coli isolates from children with diarrhea were subjected into multiplex PCR. Furthermore the strains were typed serologically with O antisera and their fliC gene was characterized by PCR-RFLP. The results obtained revealed that overall 41 (8.2%) isolates could be detected as EPEC by this multiplex PCR assay. Of these isolates; 27 (66%) were typical (escv+, bfp+) and 14 (34%) atypical EPEC (escv+, bfp-). None of these 41 isolates contained the Stx1 and Stx2 genes. Among 37 (90%) typeable strains, nine different serogroups were present. The most common serogroups were O111, followed by O86, O55 and O119 and 10 different H types were found among these isolates. The multiplex PCR assay was found to be rapid and reliable in comparison to serological test; especially when screening the large number of isolates.     

Diarrhea caused by enterotoxigenic Escherichia coli infection of humans is inhibited by dietary calcium

BACKGROUND & AIMS: In several rat infection experiments, we have shown that dietary calcium inhibits intestinal colonization and translocation of invasive salmonella. The aim of the present study was to find out whether calcium is also protective against enterotoxigenic Escherichia coli (ETEC) infection. This was first tested in our rat model and subsequently verified in a human infection study. METHODS: Rats were fed a purified diet with either a low or a high amount of calcium phosphate and orally infected with ETEC. In addition, a parallel, double-blind, placebo-controlled intervention study of 3 weeks was performed with 32 healthy men. Subjects largely maintained their habitual diet and consumed either regular milk products (calcium supply, 1100 mg/day) or placebo milk products (calcium supply, 60 mg/day). On day 10, subjects ingested a live but attenuated ETEC strain (strain E1392/75-2A), able to induce mild although short-lived symptoms. Primary outcomes studied were infection-induced diarrhea (total fecal output and relative fecal dry weight) and fecal mucin excretion. RESULTS: In humans, ETEC induced diarrhea in both groups, in that total fecal output doubled and mean relative fecal dry weight dropped from 25% to 20%. Additionally, fecal mucin excretion was increased in both groups. All these fecal parameters were completely normalized in the calcium group on the second infection day, in contrast to the placebo group, which recovered on the third infection day. Likewise, supplemental calcium inhibited ETEC colonization and diarrhea in rats. CONCLUSIONS: Calcium in milk products improves human resistance to ETEC infection as it inhibits infectious diarrhea.     

Immunogenic Evaluation of Bivalent Vaccine Candidate against Enterohemorrhagic and Enterotoxigenic Escherichia coli

Background: Caused by bacterial, viral, and parasitic pathogens, diarrhea is the second leading cause of death among children under five. Two strains of E. coli, namely Enterotoxigenic, ETEC and Enterohemorrhagic EHEC are the most important causes of this disease in developing countries. EHEC is a major causative agent of bloody diarrhea and hemorrhagic uremic syndrome, while ETEC is the most important cause of diarrhea in neonates and travelers. Objective: To evaluate the immunologic properties of a subunit vaccine candidate comprising the main immunogenic epitopes from these two bacterial strains. Methods: The construct comprised of LTB and CfaB antigens from ETEC, and Intimin and Stx2B antigens from EHEC, was designed, analyzed and synthesized using bioinformatics methods. The chimeric gene was sub-cloned in the expression vector and expressed in E. coli host. The purified chimera protein was injected subcutaneously into the experimental animals. The production of specific antibodies was confirmed by immunological methods, and the protection capacity was evaluated by the challenge of immunized mice with the pathogenic bacteria. Results: Chimeric recombinant protein was able to increase IgG titer. Neutralization assay indicated that the antibodies generated against LtB moiety were able to neutralize ETEC toxin. In animal challenge study, all non-immune mice died within 3 days after the injection of toxin, but all immunized mice survived from Stx toxin. Conclusion: The immunity to both ETEC and EHEC bacteria is significant, and this structure can be considered as a candidate for vaccine production against these bacterial strains.     

A Waterborne Outbreak of Gastroenteritis in the Golan Heights due to Enterotoxigenic Escherichia coli

Summary Background: Over a period of 4 days between May 18–21, 1998, a multifocal outbreak of diarrhea, involving 175 Israel Defence Force soldiers and at least 54 civilians, occurred in the Golan Heights. Patients and Methods: Stool samples from 40 affected soldiers were collected for microbiological testing. In addition, a rapid PCR technique was employed for the direct detection of the heat-labile (LT) and heat-stable toxin (ST) genes of enterotoxigenic Escherichia coli (ETEC) in stool samples. Results: All 40 stool specimens take from patients with diarrhea was negative by culture. However, ETEC was detected in 16 stool specimens using the rapid PCR method. The epidemiological investigation found no association between the food items consumed prior to the onset of the outbreak and the attack rate of diarrhea. A review of the water distribution system revealed that all affected military posts and civilian communities were supplied by a common water pipeline. Water sampled from various points along the distribution system showed inadequate chlorination and high concentrations of E. coli. Conclusion: This report suggests that the involvement of ETEC in the etiology of waterborne diarrheal outbreaks may be underestimated, probably due to the difficulties involved in the laboratory identification of this enteropathogen. Adoption of our rapid method for the identification of ETEC, which is applicable to routing diagnostic laboratories, facilitates pathogen detection within hours, and allows early intervention in cases of widespread diarrheal epidemics. Received: August 20, 1999 · Revision accepted: June 30, 2000