实时荧光PCR快速检测粪便中艰难梭菌方法

目的:建立实时荧光PCR快速检测艰难梭菌的方法。方法:以艰难梭菌磷酸丙糖异构酶(tpi)基因的保守序列为模板设计和合成特异性引物和荧光标记探针,建立实时荧光PCR检测体系,通过检测含有艰难梭菌标准菌株浓度为106-10 CFU/ml的细菌培养物及加标模拟样本进行敏感性分析,并对其特异性和干扰性进行评价。结果:该方法只对艰难梭菌进行特异性扩增,其他常见的病原菌均不能扩增;整个检测过程只需要2 h,对艰难梭菌菌悬液可检测至10 CFU/ml细菌,对加标粪便样本可检测至1000 CFU/ml细菌。结论:本研究建立的实时荧光PCR检测艰难梭菌方法具有快速、特异、敏感性高等优点,能实现对艰难梭菌的快速检测。     

Taqman实时定量PCR检测产毒艰难梭菌方法的建立

目的 建立以毒素基因A/B为靶基因的产毒艰难梭菌的快速定量检测方法.方法 通过设计艰难梭菌毒素A/B基因的特异引物及探针,建立标准产毒菌株DNA(ng)含量与Ct值的标准曲线.结果 该方法仅对产毒艰难梭菌进行特异性扩增,11种其他常见的致病菌及非产毒艰难梭菌均不能扩增; tcdA和tcdB基因扩增标准曲线线性关系R值分别为0.9975、0.9984,检测低限均为2.5×10-3ng.结论 该研究建立的方法具有快速、灵敏、特异性高等优点,可用于艰难梭菌毒素基因的定量检测.     

中国部分地区艰难梭菌PCR 核糖体分型及毒素基因多态性研究

目的了解中国部分地区艰难梭菌聚合酶链反应（PCR） 核糖体型别分布及其A、B毒素基因多态性,为建立适宜中国的艰难梭菌分子检测和分子分型技术提供基础数据,同时在基因水平上为艰难梭菌感染导致的复杂临床表现提供依据。方法对中国3个城市（北京、广州、济南）分离的64株艰难梭菌临床株进行PCR 核糖体分型,并对不同型别的26株代表菌株的A、B毒素基因进行扩增测序。结果64株艰难梭菌中,毒素基因型以A+B+型（45株,70.31%）为主,A-B+型19株（29.69%）。共存在9种PCR 核糖体型别,以017型（21株,32.81%）为主要型别,其次为001型（13株,20.31%）、012型（11株,17.19%）。A-B+菌株中,14株(73.68%)是017型,1株是001型。A、B毒素基因呈现一定的多态性,其中有7种A毒素序列型别（TSTA）,6种B毒素序列型别(TSTB),8种A、B毒素序列型别组合（TSTG）。结论我国部分地区的艰难梭菌可能以PCR 核糖体017型为主,A、B毒素基因在菌株间存在多态性,且核糖体型别与毒素基因多态性间存在相对应的关联。应进一步扩大菌株数量和范围,探寻适合我国的分子检测和分子分型方法,从而帮助医院更好地预防和控制艰难梭菌感染。     

多重PCR检测毒力型艰难梭菌的方法学研究及应用

目的建立多重PCR方法检测毒力型艰难梭菌。方法设计艰难梭菌种特异tpi引物和毒力基因tcdA、tcdB特异性引物,建立多重PCR方法。利用已知菌株,验证方法的特异性和最低检出限。与厌氧培养法、ELISA法比较其检测临床菌株和其毒素分泌的准确性。结果多重PCR方法检测艰难梭菌的最低检出浓度为0.7 pg/μ1,特异性为100%。53株厌氧培养法鉴定的艰难梭菌临床分离株,多重PCR方法检测tpi基因均为阳性,其中tcdA+/tcdB+为39株,tcdA-/tcdB-为14株。ELISA法检测毒素A/B显示23株为阳性、30株为阴性。23株ELISA法毒素A/B阳性的艰难梭菌多重PCR方法检测结果均为tcdA+/tcdB+。结论多重PCR方法可用于检测毒力型艰难梭菌具有较高的临床应用价值。     

葡萄球菌肠毒素B基因的PCR快速检测法

目的 建立快速检测葡萄球菌肠毒素B(staphylococcal enterotoxin B,SEB)基因的聚合酶链反应(PCR)方法.方法 ①根据SEB基因的序列,设计PCR引物特异性扩增靶基因片段.通过对1株产SEB金黄色葡萄球菌和9株对照菌株进行PCR检测,评价该方法的特异性;通过对产SEB金黄色葡萄球菌菌株做10倍系列稀释后进行PCR检测,评价该方法的敏感性;②分析30株金黄色葡萄球菌SEB基因的携带情况.结果 ①建立PCR方法快速检测SEB基因,扩增产物长度为494 bp.PCR反应体系中有26 CFU的产SEB金黄色葡萄球菌即可检出SEB基因;②30株分离的金黄色葡萄球菌中有2株携带有SEB基因.结论 PCR法可以快速、敏感地检测SEB基因,为金黄色葡萄球菌食物中毒诊断提供依据.     

合肥市屠宰生猪肉样沙门菌的PCR快速检测

目的:了解合肥市屠宰生猪肉样中沙门菌的污染状况。方法:应用聚合酶链式反应(polym erase chain reaction,PCR)对200份生猪肉样进行沙门菌的快速检测,并与常规法检测结果以及国内其他11个城市生猪肉样沙门菌的检出率作比较分析。结果:200份生猪肉样中沙门菌检出率为20%(40/200),污染率处于较高水平,而常规法的检出率为13%(26/200)。结论:合肥市生猪肉样中沙门菌的污染状况较为严重,存在发生食源性沙门菌病的潜在危险,PCR技术对沙门菌检测时间仅需2 d,与常规法相比,快速、简便、准确的优势特点使其成为调查食源性沙门菌的有用工具。     

使用Real-Time PCR粪便中检测艰难梭状芽孢杆菌

目的：探讨Real-TimePCR对粪便检测艰难梭菌的可行性。方法：以组织培养细胞毒素试验为标准，参考艰难梭菌的毒素A／B基因相应的引物和分子信标探针，对标准菌株和粪便提取的DNA实行Real-TimePCR扩增。结果：Real-TimePCR的敏感度为96．9％；特异性为100％；阳性预测值为100％；阴性预测值为98．3％。结论：Real—TimePCR与传统诊断方法相比具有快速、敏感、特异等优点，可以提高艰难梭菌的检测水平。     

多重聚合酶链反应方法检测艰难梭菌毒素基因研究

目的探讨同时检测艰难梭菌毒素A、B基因和二元毒素基因的多重聚合酶链反应(PCR)方法。方法收集2014年1-9月医院120例住院腹泻患者的粪便标本,用艰难梭菌选择性培养基CCFA分离培养艰难梭菌;采用法国生物梅里埃公司厌氧菌鉴定试剂盒鉴定;采用酶免夹心与终点荧光检测相结合技术(ELFA),检测艰难梭菌毒素A、B基因,并分析其毒素特征;将艰难梭菌纯培养出的菌落用多重PCR方法测定艰难梭菌的毒素A、B基因和二元毒素基因。结果 120例腹泻患者送检粪便标本中,分离培养出19株艰难梭菌,艰难梭菌分离率达15.8%;应用免疫学方法检测毒素检出率为52.63%;用多重PCR方法艰难梭菌A、B毒素基因检出率为94.7%;15例患者送检粪便标本检测艰难梭菌A、B毒素阳性中,10株分离培养并鉴定出艰难梭菌;105例患者送检粪便标本检测艰难梭菌A、B毒素阴性中,有9例分离培养并鉴定出艰难梭菌。结论利用多重PCR检测艰难梭菌毒素A、B基因和二元毒素基因,较传统的免疫学方法敏感性高,能够准确地区分艰难梭菌毒素基因的类型,多重PCR是一种快速、特异的一步筛选艰难梭菌毒素基因的方法。     

Laboratory-based surveillance for vancomycin-resistant enterococci: utility of screening stool specimens submitted for Clostridium difficile toxin assay.

OBJECTIVE: To study vancomycin-resistant enterococci (VRE) gastrointestinal colonization prevalence in high-risk hospitalized patients and to assess the cost and utility of this laboratory-based surveillance. SETTING: Large university teaching hospital. DESIGN: Quarterly prevalence culture survey of 50 stool specimens submitted for Clostridium difficile toxin A assay from October 1996 through June 1999 (n=526). Screening culture survey of all C difficile-positive stool specimens from July 1998 through June 1999 (n=140). PATIENTS: Specimens for analysis were collected from patients who were admitted to the hospital and who had C difficile toxin A testing ordered. Patient samples were excluded from analysis if they were obtained from patients not hospitalized at UCLA Medical Center, if the C difficile toxin assay result was indeterminate, or if the patient was known to have previous VRE colonization or infection. RESULTS: During quarterly surveillance, VRE was detected in 19.8%, C difficile toxin A in 9.5%, and both VRE and C difficile toxin A in 3.2% of stool specimens submitted for C difficile toxin assay. Patients whose stool specimens were positive for C difficile toxin A were significantly more likely than those whose specimens were negative to have VRE detected (odds ratio, 2.3; 95% confidence interval, 1.2-4.5). Based on these findings, in July 1998, we began routine screening of all C difficile-positive stool specimens for VRE. From July 1998 through June 1999, 58 (41.4%) of 140 patients with C difficile-positive specimens had VRE newly detected in the stool. The combined cost of the two laboratory-based surveillance strategies was approximately $62 per VRE-positive patient identified and $5,800 per year. CONCLUSION: Quarterly surveillance of stool submitted for C difficile assay combined with screening all C difficile-positive stools is a cost-effective and efficient strategy for detecting VRE stool colonization among high-risk hospitalized patients. Such a laboratory-based surveillance should be included as part of a comprehensive program to limit nosocomial VRE transmission.     

快速、准确的B族链球菌检测——即时荧光探针核酸扩增技术的应用

目的 研究和建立即时荧光探针核酸扩增技术,即荧光PCR检测方法,探讨B族链球菌检测的临床应用和性能.方法 首先采用标准化荧光PCR技术和操作流程,进行分析性灵敏性和特异性的鉴定,再收集临床送检样品做进一步认证.结果 分析性灵敏性达到1×102 copies/ml,分析性特异性达到特定靶序列设计要求,标准B族链球菌检测呈阳性,其他生殖道和直肠部位的易感菌株12株均阴性.并具备高重复性;以B族链球菌培养方法做对比,收集1 223例临床样本,实验结果显示:荧光PCR阳性检出率为17.58％,传统培养方法为10.87％.对PCR(+)培养(-)的72例,再扩增并测序,均检出B族链球菌的特有基因序列.结论 荧光PCR检测技术,检测B族链球菌具有较高的灵敏性和特异性,特别有助于孕妇临产前快速检测的应用.     

The role of glutamate dehydrogenase for the detection of Clostridium difficile in faecal samples: a meta-analysis

Clostridium difficile causes a serious, occasionally fatal, hospital-acquired infection. The laboratory diagnosis of C. difficile infection (CDI) needs to be accurate to ensure optimal patient management, infection control and reliable surveillance. Commercial enzyme-linked immunosorbent assays for C. difficile toxins have poor sensitivity when compared with cell culture cytotoxin assay (CTA) and toxigenic culture (TC). We performed a meta-analysis of the role of glutamate dehydrogenase (GDH) in diagnosis of CDI. We analysed 21 papers, of which eight were excluded. We included publications of original research that used a 'gold standard' reference test (either CTA or TC). We also included publications that used culture without toxin testing of the isolate as a reference test even though this is not recognised as a gold standard. Exclusion criteria were failure to use a gold standard reference test and where the index test was used as the gold standard. Significant heterogeneity between study results justified the summary receiver operating characteristic (SROC) analysis. The meta-analysis demonstrated high diagnostic accuracy of GDH for the presence of C. difficile in faeces; when compared with culture it achieved a sensitivity and specificity of >90%. The SROC plot confirmed this finding. As a surrogate for toxigenic strains the GDH yields a specificity of 80-100% with a false positivity rate of ～20%, as it detects toxigenic and non-toxigenic strains of the organism. However, GDH test has high sensitivity and negative predictive value and would be a powerful test in a dual testing algorithm when combined with a test to detect toxin.     

应用Taqman实时PCR法检测猪肉中单核细胞增生李斯特菌

目的建立敏感快速的检测猪肉中单核细胞增生李斯特菌的实时PCR方法。方法以hlyA基因为靶标,建立并验证实时PCR法的特异性。选用单核细胞增生李斯特菌CMCC 54004,制备不同浓度的纯菌液,用实时PCR进行检测,制作标准曲线并计算扩增效率。进行人工染菌实验,染菌量分别为每25g猪肉样本1.3×100、1.3×101、1.3×102、1.3×103、1.3×104、1.3×105和1.3×106CFU。分别在增菌0、4、8、12、18、24、30、36和46 h取1 ml培养液,提取DNA进行实时PCR检测,并用PCR和传统方法进行检测,比较3种方法检测的敏感性和特异性。采集24份市售猪肉样本,用这3种方法进行检测,进一步比较三者的阳性检出率。结果建立的实时PCR法特异性好,对纯菌液的检出限为1.3×103CFU/ml。人工染菌样本增菌24 h后,实时PCR检出限1.3 CFU/25 g,PCR及传统方法达到这一检出限需要增菌46 h。根据增菌24 h的检测结果,建立实时PCR样本标准曲线。24份猪肉样本,实时PCR检出17份阳性,阳性率70.83%(17/24),与PCR和传统方法的阳性率一致。根据所得的样本标准曲线,对检测样本进行定量分析,确定了阳性样本中初始含量菌。结论所建立的实时PCR具有快速简便、敏感特异等优点,整个操作可在27 h内完成,适用于猪肉中单核细胞增生李斯特菌的快速定量检测。