实时荧光定量PCR检测沙门菌flor基因和sul2基因方法的建立

目的 建立一种flor基因和sul2基因的SYBR Green Ⅰ实时荧光定量PCR方法,为检测沙门菌的耐药性以及耐药基因分子流行病学调查奠定基础.方法 以氟苯尼考、复方磺胺甲噁唑抗性的沙门菌菌株为材料,根据GenBank中登陆的沙门菌flor和sul2基因序列,设计并合成引物,建立基于SYBR Green Ⅰ染料技术的Real-Time PCR检测体系,绘制出标准曲线并对其溶解曲线进行分析.结果 以pMD 18-T为载体构建的标准品的Ct值与样品的浓度的对数在一定范围内呈现良好的线性关系,两基因的检测域值较小,flor基因可以检测到102 copies/μL的样品,sul2基因可以检测到103 copies/μL的样品,经耐药性与基因表达量相关分析,耐药性较高的菌株其flor基因、sul2基因表达量也较高.结论 建立的检测沙门菌中flor因和sul2基因的荧光定量PCR方法具有很好的灵敏性、特异性和重复性,可以广泛用于临床中lor和sul2两种基因的检测以及沙门菌耐药分子流行病学的调查.     

荧光定量PCR检测重组人干扰素α2b中宿主基因组的残留DNA

目的 采用荧光定量PCR法检测注射用重组人干扰素α2b半成品中宿主基因组的残留DNA.方法 选择重组人干扰素α2b工程菌的宿主菌23S核糖体RNA基因为模板设计扩增引物,建立基于SYBR Green Ⅰ荧光染料的荧光定量PCR检测法.结果 宿主基因组DNA的量与荧光定量反应的Ct(循环阈值)值呈良好的线性关系(r~2=0.9803).结论 所用方法操作简便、快速,可用于重组人干扰素α2b制备过程中的质量监控及半成品的检定.     

Taqman荧光定量PCR法在药品沙门菌快速检测中的应用

目的：建立药品中沙门菌的荧光定量PCR快速检测方法。方法：根据沙门菌的特异基因序列合成引物和探针,提取沙门菌DNA进行检测。结果：该方法特异性好,灵敏度达到160cfu·ml-1,人工污染的药品检出率为100％．结论：荧光定量PCR法可用于药品沙门菌的快速检测。     

巢式聚合酶链反应快速检测血液标本中

目的建立一种检测血液中伤寒沙门菌的方法.方法根据伤寒沙门菌特异鞭毛基因设计两对引物,通过巢式聚合酶链反应(PCR)扩增伤寒沙门菌DNA片断,扩增产物通过凝胶电泳进行分析.结果用伤寒沙门菌DNA系列稀释液进行试验,巢式PCR能够检出10个伤寒沙门菌.36份培养阳性标本,33份PCR阳性;6份培养阴性但临床高度可疑的伤寒病人标本PCR阳性10份其它原因引起发热的临床标本均为阴性.结论巢式PCR能够快速、特异、准确地检出血液中的伤寒沙门菌.     

巢式聚合酶链反应快速检测血液标本中伤寒沙门菌的实验研究

目的 建立一种检测血液中伤寒沙门菌的方法。方法 根据伤寒沙门菌特异鞭毛基因设计两对引物,通过巢式聚合酶链反应（PCR）扩增伤寒沙门DNA片断,扩增产物通过凝胶电泳进行分析。结果 用伤寒沙门菌DNA系列稀释进行试验,巢式PCR能够检出10个伤寒沙门菌。36份培养阳性标本,33份PCR阳性;6份培养阴性但临床高度可疑的伤寒病人标本PCR阳性;10份其它原因引起发热的临床标本均为阴性。结论 巢式PCR能够快速、特异、准确地检出血液中的伤寒沙门菌。     

荧光PCR法与细菌培养法在鼠伤寒沙门菌食物中毒检测中的应用比较

目的把荧光PCR法与细菌培养法在鼠伤寒沙门菌食物中毒检测中的应用进行比较。方法使用以往的细菌培养方式和荧光PCR方式对10份样本实施检验。结果这10份样本经过检验之后,PVR检验出沙门菌5株,阳性概率相对较高为50%,以往的方式检验出3株沙门菌,阳性概率为30%相对较低。结论与以往的培养方式进行对比,使用PCR方式能够增强检验出鼠伤寒沙门菌的概率,节约检验时长,合适用在食物中毒这一方面的检验。     

SYBR GreenⅠ实时荧光PCR检测VKORC1和CYP2C9基因型

目的建立一种检测维生素K环氧化物还原酶复合体亚单位1(VKORC1)和细胞色素P450 2C9(CYP2C9)基因型的SYBR GreenⅠ实时荧光PCR法。方法提取134例心脏瓣膜置换术患者的外周血基因组DNA。针对VKORC1 1173C>T和CYP2C9 1075A>C两个单核苷酸多态性(SNP)位点,分别设计一套等位基因特异性引物。PCR反应体系中加入荧光染料SYBR GreenⅠ,样本经扩增后结合熔解曲线分析,确定每个样本的基因型。结果 VKORC1 1173C>T和CYP2C9 1075A>C两个SNP位点的不同基因型具有不同的熔解曲线峰型。134例患者中,VKORC1 1173C>T基因型TT、TC和CC分别有111例、21例和2例,各占82.8%、15.7%和1.5%;CYP2C9 1075A>C基因型*1/*1和*1/*3分别有121例和13例,各占90.3%和9.7%,未检出纯合子*3/*3基因型。结论基于荧光染料SYBR GreenⅠ的实时荧光PCR分型方法操作简便、价格低廉、结果准确可靠,适于临床实验室对VKORC1 1173C>T和CYP2C9 1075A>C的基因分型。     

伤寒、甲、乙、丙型副伤寒沙门菌多重荧光PCR方法的评估及临床应用

目的建立和评估多重荧光PCR方法快速鉴定伤寒、甲、乙、丙型副伤寒沙门菌的方法,并应用于临床血培养样品的检测。方法收集留取医院的可疑伤寒患者的血培养标本,用多重实时荧光PCR法和传统分离培养法同时进行分离鉴定,分析比较双盲实验结果,评价多重实时荧光PCR方法的灵敏度、特异度等检测性能指标。结果共检测临床样本538例,多重荧光PCR法检出阳性218例,比传统培养法多检出6例;阴性320例,与传统培养法一致。以传统检测方法为金标准,多重荧光PCR方法的灵敏度为100%,特异度为98.2%。结论建立的多重荧光PCR检测方法操作简便,可快速、特异、灵敏地检测出伤寒、甲、乙、丙型副伤寒沙门菌,可以应用于临床标本的早期快速分型诊断,提升重大传染病的应急处置能力和监测能力。     

SYBR Green I实时荧光PCR检测VTKORC1和CYP2C9基因型

目的 建立一种检测维生素K环氧化物还原酶复合体亚单位1(VKORC1)和细胞色素P450 2C9(CYP2C9)基因型的SYBR GreenI实时荧光PCR法.方法 提取134例心脏瓣膜置换术患者的外周血基因组DNA.针对VKORC11173C>T和CYP2C9 1075A>C两个单核苷酸多态性(SNP)位点,分别设计一套等位基因特异性引物.PCR反应体系中加入荧光染料SYBR Green I,样本经扩增后结合熔解曲线分析,确定每个样本的基因型.结果 VKORC1 1173C>T和CYP2C9 1075A>C两个SNP位点的不同基因型具有不同的熔解曲线峰型.134例患者中,VKORC1 1173C>T基因型TT、TC和CC分别有111例、21例和2例,各占82.8%、15.7%和1.5%;CYP2C9 1075A>C基因型*1/*1和*1/*3分别有121例和13例,各占90.3%和9.7%,未检出纯合子*3/*3基因型.结论 基于荧光染料SYBR Green I的实时荧光PCR分型方法操作简便、价格低廉、结果准确可靠,适于临床实验室对VKORC1 1173C>T和CYP2C9 1075A>C的基因分型.     

实时荧光定量PCR方法应用于药品无菌快速检测的研究

目的 建立应用实时荧光定量PCR进行无菌快速检测的方法。方法 选取金黄色葡萄球菌及大肠埃希菌,用裂解试剂盒抽提细菌基因组DNA,进行实时荧光定量PCR检测,并应用叠氮溴化丙锭（PMA）抑制样品中死菌基因组DNA的PCR扩增。结果 PMA能有效去除样品中死菌干扰,针对16S rRNA基因保守序列进行扩增的荧光定量PCR方法具有较高的灵敏度。在金黄色葡萄球菌和大肠埃希氏菌检测中,最低含菌量组与阴性对照组Ct值有明显差异（P〈0.05）,其最低检出限为2 CFU/PCR。在对人工污染药品的无菌检测中,该方法与药典检测方法结果一致。结论 进行无菌检查时,采用PMA去除样品中死菌基因组DNA干扰,以裂解试剂盒抽提细菌基因组后用荧光定量PCR分析样品中细菌污染,可将检测时间缩短到4 h左右,操作简单,灵敏性高,可应用于药品无菌检查的快速筛查。     

PCR detection of Salmonella typhimurium in pharmaceutical raw materials and products contaminated with a mixed bacterial culture using the BAX system

The BAX system, a PCR-based assay, was evaluated for detecting Salmonella typhimurium in pharmaceutical raw materials and products contaminated with mixed bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella typhimurium. Artificially contaminated samples were preenriched in lactose broth with and without Tween 20. After preenrichment, samples were analyzed by PCR and standard methods. Ten of 25 samples did not show presence of the specific Salmonella spp. 740-base pair DNA fragment. However, S. typhimurium was isolated and identified by standard methods from all 25 samples. To optimize S. typhimurium detection in PCR negative samples, lactose broth was replaced by buffered peptone water (BPW) as the preenrichment broth. When BPW was used, all 10 samples were PCR positive. BPW enrichments increased S. typhimurium growth resulting in rapid PCR detection. The presence of non-Salmonella bacteria influenced the performance of the PCR-based assay. Optimization of S. typhimurium PCR detection in mixed culture required the use of different preenrichment broths. However, the BAX system detected S. typhimurium within 27 hours while standard methods required 5-7 days.     

二重实时PCR快速检测耐甲氧西林金黄色葡萄球菌

目的 建立二重实时聚合酶链反应（duplexreal-timePCR）快速检测耐甲氧西林金黄色葡萄球菌（MRSA）。方法 采用二重SYBR Green实时PCR快速检测MRSA的决定基因mecA和金葡菌的种特异性基因nuc，经熔解曲线分析鉴定产物。结果 所有MRSA菌株的熔解曲线均呈现mecA、nuc基因特异性的峰，甲氧西林敏感的金葡菌仅有nuc峰，耐甲氧西林的表葡菌仅有mecA峰，甲氧西林敏感的表葡菌与其他菌种的菌株无特异峰出现；当MRSA菌浓度达10^2cfu／ml时就可检出。在131株金葡菌中，30．53％（40／131）耐药；二重实时PCR扩增mecA基因29．01％（38／131）阳性，nuc基因100％阳性。单引物与二重实时PCR两者阳性扩增符合率为100％。结论 对mecA、nuc基因进行二重实时PCR能准确、快速地鉴定耐甲氧西林的金葡菌和凝固酶阴性的葡萄球菌。     

Detection of decreased susceptibility to fluoroquinolones in Salmonella spp. by five different methods including real-time polymerase chain reaction (PCR)

Detection of Salmonella spp. isolates showing decreased susceptibility to fluoroquinolones has become important owing to the increasing prevalence of these strains and their association with treatment failure. Nalidixic acid agar dilution, nalidixic acid disk diffusion, MicroScan automated system and real-time polymerase chain reaction (PCR) (LightCycler) followed by melting temperature (Tm) analysis are compared with ciprofloxacin agar dilution as suitable methods to detect decreased susceptibility to fluoroquinolones in 100 Salmonella spp. isolates. Three minor discrepancies were found for nalidixic acid disk diffusion, one minor discrepancy was found for nalidixic acid agar dilution and Tm analysis, and one major discrepancy was found for MicroScan. Nalidixic acid disk diffusion was confirmed as a good screening method. Tm analysis is a rapid and accurate method for detecting decreased susceptibility to fluoroquinolones due to gyrA mutations in Salmonella spp.     

多重PCR技术检测沙门菌两种四环素耐药基因

应用两对特异性引物同时检测四环素耐药基因tetB和tetC通过对35株沙门菌分离株的四环素耐药性检测，表明所有沙门菌分离株均含tetC基因，与药敏试验结果阳性符合率65．7％，8株同时含有tetB基因，与药敏试验结果阳性符合率100％，tetB基因和tetC基因双阳性的菌株与药敏试验结果阳性符合率也为100％。取其中部分菌株扩增出tetB和tetC基因片段进行序列分析，5株菌的tetB基因扩增产物序列完全相同，与质粒pRT11相应序列同源性达99．7％；14株菌的tetC基因扩增产物与质粒pBR322中的相应序列同源性为100％。证实沙门菌耐药基因普遍存在，且同时含有tetB和tetC基因的菌株表现耐药。多重PCR技术同时检测两种四环素耐药基因，适合大量样本的检测，对开展沙门菌四环素多种耐药基因的分子流行病学监测提供了有效途径。     

实时荧光定量PCR检测ESBLs基因型方法的研究

目的:建立一种快速检测革兰阴性杆菌产超广谱β内酰胺酶(ESBLs )耐药基因分型的SYBR GREEN Ⅰ实时荧光定量PCR方法.方法:针对临床常见ESBLs的耐药基因SHV、TEM、 CTX-M、OXA及其同源性分析,设计了SHV、TEM 、CTX-M-1、CTX-M-2、CTX-M-8、CTX-M-9、OXA-1、OXA-2及OXA-10 共9对特异性引物,煮沸法提取DNA模板,建立并优化SYBR GREEN I实时荧光定量PCR反应体系,并对其精密度、线性范围进行测定.利用建立方法对51株表型阴性的多重耐药的大肠埃希菌、肺炎克雷伯菌进行ESBLs耐药基因检测,并与改良三维实验进行对比.结果:从39株ESBLs表型阳性菌株及51株ESBLs表型阴性多重耐药菌株中扩增出除OXA-2外共8种耐药基因型并经测序证实.线性检测范围3×10~3~3×10~8拷贝/mL , r ＝ -0.994 7 ;批间重复性试验变异系数(CV)为9.6%.荧光定量PCR方法与改良三维实验方法比较差异无统计学意义( χ2 = 1.125,P > 0.05).结论:SYBR GREEN Ⅰ实时荧光定量PCR检测ESBLs的耐药基因具有特异性强、灵敏度高、快速、简便的特点,适于临床监测革兰阴性杆菌产ESBLs 基因型.     

Designing a biochip following multiplex polymerase chain reaction for the detection of Salmonella serovars Typhimurium,Enteritidis, Infantis,Hadar, and Virchow in poultry products

Salmonella-contaminated foods, especially poultry-derived foods (eggs, chicken meat), are the major source of salmonellosis. Not only in the European Union (EU), but also in the United States, Japan, and other countries, has salmonellosis been an issue of concern for food safety control agencies. In 2005, EU regulation 1003/2005 set a target for the control and reduction of five target Salmonella enterica serovars—S. Typhimurium, S. Enteritidis, S. Infantis, S. Hadar, and S. Virchow—in breeding flocks. Thus, a simple biochip for the rapid detection of any of these five Salmonella serovars in poultry products may be required. The objectives of this study were to design S. Virchow-specific primers and to develop a biochip for the simultaneous identification of all or any of these five Salmonella serovars in poultry and poultry products. Experimentally, we designed novel polymerase chain reaction (PCR) primers for the specific detection of S. Virchow, S. Infantis, and S. Hadar. The specificity of all these primers and two known primer sets for S. Typhimurium and S. Enteritidis was then confirmed under the same PCR conditions using 57 target strains and 112 nontarget Salmonella strains as well as 103 non-Salmonella strains. Following multiplex PCR, strains of any of these five Salmonella serovars could be detected by a chromogenic biochip deployed with DNA probes specific to these five Salmonella serovars. In comparison with the multiplex PCR methods, the biochip assay could improve the detection limit of each of the Salmonella serovars from N × 10³ cfu/mL to N × 10² cfu/mL sample in either the pure culture or the chicken meat samples. With an 8-hour enrichment step, the detection limit could reach up to N × 10⁰ cfu/mL.     

Identification of bacteria in juice/lettuce using magnetic nanoparticles and selected reaction monitoring mass spectrometry

Ensuring food safety requires a rapid and reliable method for detecting food-borne pathogens. Mass spectrometry has been demonstrated as a powerful tool to classify pure bacterial species. However, matrix interference from food backgrounds may lead to false results because of the suppression of microbial signals. It is useful to develop a method for bacterial enrichment and marker identification in food samples. Magnetic zirconia nanoparticles were used to concentrate spiked microorganisms from apple juice/lettuce under specific conditions (pH 4.5). Bacterial identification was achieved using nanoLC–MS. Selected reaction monitoring of bacteria-related peptides was applied for the first time to identify bacteria including Staphylococcus aureus and Escherichia coli. This study presents an accurate means for bacterial identification in food matrixes using MS. The analysis time is less than 90 min and the minimum concentration of E. coli detected was 5 × 10³ CFU/mL. The interaction between bacteria and the magnetic nanoparticles was electrostatic and nonspecific, in contrast to immunoassays which require specific antibodies. The targeted peptide analysis focuses on the bacterial markers, thus significantly simplifying the analysis and leading to an accurate identification of bacteria.     

免标准曲线定量PCR技术的探讨

目的实际应用发现,传统的定量PCR方法误差较大不能满足科学研究以及临床精确定量的需要,本文拟探讨如何选择分析使用的技术资料以降低分析误差以及分析判断误差的主要来源。方法使用PCR动力学模型模拟实时定量PCR仪器跟踪测定资料,使用不同扩增时期的荧光数值资料计算起始模板数、分析误差,并根据仪器的荧光信号测定误差随机对资料加入误差后,分析结果的误差与研究误差的分布。结果使用指数扩增期内后期的5个资料点分析,可以获得当前仪器误差水平下,相对误差最小的结果。使用的资料点一旦包含有一个线性扩增期资料,结果的误差即显著增加。该文亦给出了一般性最后一次指数扩增循环的判定方法。结论免标准曲线的定量PCR方法将成为今后定量PCR的主要技术手段。     

单管半套式PCR-SSCP用于结核分支杆菌rpoB基因突变的研究

目的 ①证明单管半套式聚合酶链反应(PCR)是检测结核分支杆菌的特异性方法;②研究结核分支杆菌rpoB基因突变与利福平耐药性的关系。方法 设计三条针对结核分支杆菌的特异性引物,采用单管半套式PCR-单链构象多态性(SSCP)方法对结核分支杆菌、其它分支杆菌和富含GC碱基的细菌进行分析。结果 ①结核分支杆菌标准菌株H_(37)Rv和55株临床分离株均扩增出一条193bp片段,其特异性为100％,敏感性约为10pgDNA;15株非结核分支杆菌和8株富含GC的细菌均未得到扩增;②55株结核分支杆菌临床分离株193bp扩增片段SSCP图谱特点:以H_(37)Rv为对照,15株利福平敏感株均无区别;40株耐药株除6株外其余34株SSCP图谱有明显区别,检测阳性率为85％。结论 单管半套式PCR检测结核分支杆菌简便、敏感、特异,结合SSCP可检出耐利福平结核分枝杆菌rpoB基因突变,此突变与利福平耐药关系密切。