陕西省E.coli O157宿主动物带菌情况调查分析

E.coli O157:H7自1982年首次在美国发现以来,在世界范围内已发生过多次食源性暴发。由于该菌可致人类腹泻、出血性结肠炎和溶血性尿毒综合征,发生溶血性尿毒综合征之后死亡率极高,而引起世界普遍关注。近些年,许多地区开展了E.     

肠出血性大肠杆菌O157：H7的分子生物学检测方法研究进展

肠出血性大肠杆菌O157：H7是一种新型人类病原菌，由它引起的感染严重威胁着人类健康和生命。对其进行分子流行病学调查，可为该病的预防、诊断和治疗提供有力的依据和参考。现就目前常用的几种大肠杆菌O157：H7的分子生物学检测技术作一简介。     

酶标记鸡卵黄抗E.coli O157:H7抗体的制备与特性研究

目的：研究制备特异性抗E.coli O157：H7鸡卵黄免疫球蛋白（IgY）及酶标记抗体（IgY-HRP）的工艺条件。方法：分别用灭活菌体、脂多糖及O抗原多糖抗原免疫临产母鸡,水稀释法结合离子交换色谱分离提取IgY,再用过碘酸钠法和戊二醛法进行HRP标记,各种制品的分析用电泳及ELISA法。结果：可获得纯度为79．6％～85．3％的IgY,提取率74％～77％;过碘酸钠氧化法标记效果较好,IgY-HRP（1mg／ml）最高效价640。结论：免疫制备IgY方法可行,过碘酸钠法标记IgY-HRP取得良好效果。     

酶标记鸡卵黄抗E.coli O157∶H7抗体的制备与特性研究

目的研究制备特异性抗E.coliO157∶H7鸡卵黄免疫球蛋白(IgY)及酶标记抗体(IgY-HRP)的工艺条件。方法分别用灭活菌体、脂多糖及O抗原多糖抗原免疫临产母鸡,水稀释法结合离子交换色谱分离提取IgY,再用过碘酸钠法和戊二醛法进行HRP标记,各种制品的分析用电泳及ELISA法。结果可获得纯度为79.6%~85.3%的IgY,提取率74%~77%;过碘酸钠氧化法标记效果较好,IgY-HRP(1mg/ml)最高效价640。结论免疫制备IgY方法可行,过碘酸钠法标记IgY-HRP取得良好效果。     

大肠杆菌O157∶H7抗菌药物敏感性研究

为了解E .ColiO15 7∶H7对抗菌药物的敏感性及其耐药谱的差异 ,我们选择了常见的 19种抗生素 ,对 1999年分离到的 89株O15 7∶H7进行药敏试验。结果显示O15 7∶H7对氨基甙类、头孢类、喹诺酮类、呋喃类的实验药物、多肽类的多粘菌素B以及氯霉素的敏感率高 ,基本在 90 %以上。在宿主动物中家禽类的菌株耐药率大于家畜类 ,家畜类宿主中猪的菌株耐药率大于牛、羊。无毒力基因菌株对抗生素的耐药率高于有毒力基因的菌株 (P <0 0 5 )。样本来源不同和基因型别不同的耐药结果均显示菌株对抗生素的耐药情况与其是否携带有毒力基因有关。     

大肠杆菌O157∶H7抗菌药物敏感性研究

为了解E .ColiO15 7∶H7对抗菌药物的敏感性及其耐药谱的差异 ,我们选择了常见的 19种抗生素 ,对 1999年分离到的 89株O15 7∶H7进行药敏试验。结果显示O15 7∶H7对氨基甙类、头孢类、喹诺酮类、呋喃类的实验药物、多肽类的多粘菌素B以及氯霉素的敏感率高 ,基本在 90 %以上。在宿主动物中家禽类的菌株耐药率大于家畜类 ,家畜类宿主中猪的菌株耐药率大于牛、羊。无毒力基因菌株对抗生素的耐药率高于有毒力基因的菌株 (P <0 0 5 )。样本来源不同和基因型别不同的耐药结果均显示菌株对抗生素的耐药情况与其是否携带有毒力基因有关。     

来自腹泻合并急性肾衰病人疫区的99株E.coli O157∶H7的鉴定结果分析

目的调查研究河南省2000年3月17日-7月6日发生的腹泻合并急性肾衰病人的致病菌。方法采集病人、外环境、家畜和家禽标本620份,采用免疫磁珠集菌法、CHROMAGAR-O157∶H7显色培养基分离及营养肉汤增菌、rfbO157、rfbO111引物多重PCR扩增等方法进行病原菌的检测。结果分离出99份E.coli O157∶H7菌株,其中rfbO157、志贺氏样毒素2(stx2)、溶血素(hlyA)、肠上皮细胞纤毛消除素(eaeA)基因和vero细胞毒性试验均阳性的有56株,其它基因组合7株,36株无毒力基因。结论99株E.coli O157∶H7在CHROMAGAR-O157∶H7显色培养基生长形态、颜色,生化反应及毒力基因表现等方面出现多样化,对于今后在制定对该菌的诊断标准、传染源的控制及细菌毒力学等方面的研究提供了依据。     

环介导等温扩增技术检测粪便幽门螺杆菌方法的建立及应用

目的:建立一种快速、简单的检测粪便幽门螺杆菌的环介导等温扩增方法(loop-medi atedisotherma lamplification,LAMP)。方法:针对幽门螺杆菌的16SrRNA基因设计4条特异性引物(2条内引物和2条外引物),优化反应条件后,对方法的敏感性、特异性进行评估,并评估检测粪便模拟标本的敏感性。结果:使用LAMP方法可以在2h内得到检测结果,加入染料后阳性结果为绿色,电泳后有明显的阶梯状条带。幽门螺杆菌的基因组DNA检测敏感性为12pg,是普通PCR的10倍,对模拟粪便标本进行检测,检测限为102CFU/mL。金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌、粪肠球菌的检测结果均为阴性。结论:本研究建立的检测幽门螺杆菌的LAMP方法敏感性高、特异性强,操作简单,不需要特殊设备,是一种很有前景的检测方法。     

分离到与O157单克隆抗体及H7血清强凝集的弗劳地枸橼酸杆菌

我们从腹泻粪便及腹泻病区塘水中分离到两株革兰氏阴性杆菌,与Ｏ１５７单克隆抗体及Ｈ７血清发生强烈凝集反应。应用同位素标记探针的原位杂交试验及ＰＣＲ技术均未检查出类志贺样毒素Ⅰ和Ⅱ（ＳＬＴ－Ⅰ,ＳＬＴ－Ⅱ）。用检测出血性大肠杆菌的ＰＣＲ试剂盒,显示阴性结果。最终用美国Ｂｉｏｌｏｇ公司生产的Ｂｉｏｌｏｇ细菌鉴定分类系统,确定为弗劳地枸橼酸杆菌。该实验结果表明,我们在进行该菌流行病调查时,尤其进行Ｏ１５７∶Ｈ７埃希氏大肠杆菌分离和鉴定时,必须辅以适当的生物化学试验,以免出现假阳性结果。     

江苏省徐州市出血性大肠埃希菌O157∶H7 感染性腹泻的监测研究

目的 系统监测肠出血性大肠埃希菌O157∶H7(O157∶H7)感染并发肾衰(HUS)、腹泻患者、宿主动物、蝇类、食品携带O157∶H7的状况,为预警监测体系建立提供参考依据。 方法 采集徐州市疫情暴发点和监测点腹泻患者粪便、蝇类和食品标本分离菌株。应用PCR技术检测志贺毒素基因stx。 结果 1999-2011年徐州市累计报告O157∶H7引起的感染性腹泻并发HUS 132例。腹泻患者带菌率1.2%、蝇类带菌率0.5%、食品带菌率1.0%、宿主动物带菌率3.1%。菌株带毒由stx1和stx2共存发展为以不带毒为主。 结论 江苏省徐州市O157∶H7感染自1999年、2000年暴发流行后,该市疫情一直稳定。腹泻患者带菌率、宿主动物带菌率、蝇类带菌率、食品带菌率及菌株带毒情况发生了改变。     

Caprine arthritis encephalitis virus detection in blood by loop-mediated isothermal amplification (LAMP) assay targeting the proviral gag region

Caprine arthritis encephalitis virus (CAEV), of the genus Lentivirus of the Retroviridae family, causes persistent disease, which is characterized by polyarthritis and mastitis in adult goats and progressive paresis (leukoencephalomyelitis) in kids. A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CAEV in blood samples. Species-specific primers amplifying the gag gene region in the provirus were used for the detection of CAEV. The LAMP assay result was obtained 30 min after incubation on a constant temperature at 63 °C in a heat block. Resulting amplicons were visualized by addition of SYBR green dye after the reaction and checked by agarose gel electrophoresis. The sensitivity of LAMP assay was evaluated by comparing the result with the nested polymerase chain reaction. Based on the experiments, the result of the assay indicated a rapid and sensitive test for the detection of CAEV.     

Application of a loop-mediated isothermal amplification method for the detection of pathogenic Leptospira

Leptospirosis is an emerging infectious disease, which is considered to be the most widespread zoonotic disease in the world. There are more than 230 known serovars in the genus Leptospira. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of pathogenic Leptospira spp. was developed and evaluated through amplification of the lipL41 gene coding for the outer membrane protein LipL41. The LAMP assay did not rely on the isolation and culture of leptospires, and no cross-reactivity was observed with other bacterial species. A SYBR Green I-based LAMP assay was also carried out for the real-time detection of DNA amplification. The lower detection limit of the LAMP assay was approximately 100 copies, which was the same as the polymerase chain reaction (PCR) and real-time PCR assays. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis of the amplified product. The LAMP assay is easy to perform and inexpensive, and so may be applied in the rapid and specific diagnosis of Leptospira.     

Loop-mediated isothermal amplification (LAMP): recent progress in research and development

Loop-mediated isothermal amplification (LAMP) is an established technology that continues to attract the attention of researchers in many fields. Research and development efforts on LAMP technology in recent years have focused on two major areas; first, the study of its clinical application as an approved in vitro diagnostics tool in Japan and certain other countries; and second, research aimed at further simplifying the LAMP test process. This review provides an overview of the status of LAMP on these two topics by summarizing research work conducted, in the main, after our previous review article.     

Detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification (LAMP) assay and serology in pediatric community-acquired pneumonia

Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for treatment with effective antimicrobial agents without delay; however, this capacity has not yet been established in clinical practice. Recently, a novel nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) has been used to rapidly diagnose various infectious diseases. In this study, we prospectively evaluated the efficacy of the LAMP assay to rapidly diagnose M. pneumoniae pneumonia in clinical practice. Three hundred sixty-eight children (median age, 3.8 years; range, 0.1–14.3 years) admitted to our hospital between April 2009 and March 2010 for community-acquired pneumonia were enrolled in this study. We obtained throat swabs on admission to detect M. pneumoniae DNA and paired serum samples on admission and at discharge to assay M. pneumoniae antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or a fourfold or greater increase in antibody titer. Overall, 46 children (12.5% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia; of these, 27 (58.7%) were aged less than 6 years. Of the aforementioned 46 children, 38 (82.6%) and 37 (80.4%) were identified by LAMP and serology, respectively. When the results of serology were taken as the standard, the sensitivity and specificity and positive and negative predictive values of the LAMP assay were 78.4%, 97.3%, 76.3%, and 97.6%, respectively. We concluded the LAMP assay may be useful for rapid diagnosis of M. pneumoniae pneumonia.     

新型环烯烃聚合物微流控芯片的设计及其在大肠埃希菌O157:H7快速检测中的应用

目的建立一种可快速检测大肠埃希菌O157:H7的新型微流控芯片方法,用于细菌的早期鉴别与诊断。方法采用激光雕刻技术制作微流控芯片,在OCA光学透明胶上雕刻1条微通道,再将上、下2层环烯烃聚合物(COP)不可逆键合,最后通过免疫荧光方法检测大肠埃希菌O157:H7。比较微流控芯片法与培养法检测结果的总体符合率。结果成功建立了一种新型、简易的微流控芯片系统,对大肠埃希菌O157:H7的检测限为10~3CFU/mL,且有良好的特异性和可重复性,与培养法的总体符合率为95%。结论新型COP塑料芯片系统可实现对大肠埃希菌O157:H7的低成本、快速检测。     

中国部分地区人群和动物中肠出血性大肠埃希菌O157：H7分布特征

自20世纪90年代以来，中国大部分省、市、自治区相继开展了人群和动物的肠出血性大肠埃希菌O157：H7病原监测。监测结果显示：①在不同的地区和时间，人群中O157：H7的感染率，带菌率不同；②动物中O157：H7的带菌率存在地区、时间及种群的差异；③人群O157：H7的感染与动物带菌存在关联。提示，加强人群和动物的病原监测、揭示O157：H7在人群和动物中的流行规律、降低动物的感染或带菌率对预防与控制人群肠出血性大肠埃希菌O157：H7的感染具有重要意义。