多重PCR快速检测3种致泻性大肠埃希菌方法的建立

目的利用多重聚合酶链反应(PCR)技术,建立一种可以同时快速检测肠致病性大肠埃希菌(EPEC)、肠侵袭性大肠埃希菌(EIEC)、肠出血性大肠埃希菌(EHEC)3种致泻性大肠埃希菌的多重PCR方法。方法根据EPEC的eae基因、EIEC的ipaH基因、EHEC的stx1基因筛选设计引物,建立多重PCR检测体系,并对PCR反应体系和条件进行优化。结果设计的3对PCR引物均能特异地扩增出相应的目的基因,该多重PCR体系能同时检测3种目的菌,特异度强。结论初步建立了一种能够同时快速检测3种致泻性大肠埃希菌的多重PCR检测方法,可用于食品安全及食物中毒事件的快速筛查。     

多重PCR快速检测金黄色葡萄球菌中氨基糖苷类抗生素耐药基因及其临床应用

目的 建立金葡菌氨基糖苷类抗生素耐药基因的多重PCR快速检测体系,了解耐药基因acc(6')-Ie+aph(2″)、aph(3')-Ⅲat ant(4')-Ia在广州地区的流行分布.方法 采用VITEK-60或PHOENIX-100微生物自动鉴定系统鉴定菌株及药敏试验,利用琼脂扩散法检测4种氨基糖苷类抗生素的耐药表型.通过优化PCR体系建立多重PCR并应用于检测金葡菌氨基糖苷类抗生素耐药基因.结果 构建了四重PCR快速检测体系, 124株金葡菌临床分离株中,acc(6')-Ie+aph(2″)、aph(3')-Ⅲa和ant(4') -Ia的检出率分别为62.1%、32.3%和1.6%;所有庆大霉素、奈替米星以及阿米卡星耐药的金葡菌菌株均检测到上述3个耐药基因中的1个或1个以上基因;74株mecA阳性菌株中72株(97.3%)检测到acc(6')-Ie+aph(2″) 基因.结论 所构建的多重PCR检测体系为临床实验室提供快速而有效的菌株耐药基因检测手段.编码AAC(6')-APH(2″)双功能酶的acc(6')-Ie+aph(2″) 基因是金葡菌最重要的氨基糖苷类抗生素耐药基因,其次为aph(3')-Ⅲa,ant(4')-Ia则少见.     

多重聚合酶链反应检测革兰阳性／阴性细菌、真菌和mecA基因的临床评价

目的：评价多重聚合酶链反应（PCR）直接检测革兰阳性／阴性细菌、真菌和耐甲氧西林葡萄球菌（MRS）的可靠性和准确性。方法：采用细菌16rRNA基因通用引物、革兰阴性细菌特异引物、全真菌通用引物和mecA基因特异引物，通过多重PCR对271份临床无菌性体液扩增检测革兰阳性／阴性细菌、真菌和MRS，同时对这些标本进行细菌、真菌培养和甲氧西林对葡萄球菌MIC测定。结果：多重PCR诊断无菌性体液体革兰阳性／阴性细菌、真菌和MRS感染的特异性、灵敏度、阳性预测值、阴性预测值和诊断效率分别为98.5％、97.0％、97.0％、99.0％、98.2％、99.1％、95.9％、95.9％、99.1％、98.5％、99.6％、100％、83.3％、100％、99.6％、100％、48.0％、100％、61.8％、71.7％。结论：多重PCR直接检测无菌性体液中革兰阳性／阴性细菌、真菌和mecA基因所致的MRS具有敏感、快速、特异、准确的优点，是一种可靠的实验诊断手段，但不能代替培养法。     

布鲁氏菌多重聚合酶链反应鉴定研究

目的　在同一体系中对布鲁氏菌6个种进行鉴定。方法　根据布鲁氏菌6个种的差异基因设计5对引物,应用多重聚合酶链反应（PCR）方法对布鲁氏菌进行鉴定。先用标准菌株建立方法,优化实验条件,然后扩增地方株进行验证。结果　除绵羊附睾种外多重引物PCR方法能将其余5个布鲁氏菌种全部鉴定出来。结论　这种方法快速准确,且对实验操作人员安全,是布鲁氏菌鉴定的辅助方法。     

多重PCR技术在Y染色体微缺失快速筛查中的应用

目的探讨多重聚合酶链反应(PCR)技术在Y染色体微缺失快速筛查中的应用价值。方法以38例健康男性和46例无(少)精症男性外周血淋巴细胞基因组DNA为模板,采用针对Y染色体无精子因子(AZF)4个亚区(AZFa～d)15个序列标签位点(STS)的多重引物进行PCR检测,并以琼脂糖凝胶电泳分析PCR产物。结果 84例受试者多重PCR检测成功率为100%,且特异性好,扩增效率高。健康男性标本可检出15个STS。患者中检出AZFc+d微缺失1例。结论多重PCR是快速筛查Y染色体微缺失的有效工具。     

Identification of human enterovirulent Escherichia coli strains by multiplex PCR

Some strains of Escherichia coli are involved in enteric infections in both adults and children. However the classical diagnostic methods can not differentiate pathogenic from nonpathogenic E. coli, because of the lack of phenotypic differences. In this study, we developed multiplex PCR in order to amplify fragments of specific virulence genes of the five main E. coli pathotypes. Fragments of the expected size were obtained using previously or newly designed primers and allowed identification of 10 virulence genes in only 5 reactions. This method was applied to the detection of pathogenic E. coli isolated from 90 patients' stools specimens during an 18-month survey. Patients were suffering from diarrhea or hemolytic uremic syndrome and in 13 cases (14.4%), an enterovirulent E. coli strain was detected. This diagnostic method could therefore represent an important technique in clinical laboratories which lack standard tests for these pathogens.     

多对型特异性引物巢式PCR检测乙型肝炎病毒基因型分析

目的采用多对型特异性引物通过巢式PCR法检测HBeAg阳性患者血清中乙肝病毒(HBV)基因型的分布情况。方法根据从前S1基因到S基因中的保守序列设计出10条内外引物,并将其中8条内引物分成A、B两组分别扩增A、B、C和D、E、F型HBV,然后将第2轮PCR的两组产物分别用3%琼脂糖进行电泳,根据PCR产物片段大小直接判定HBV基因型。用该法检测15例慢性乙肝患者血清中HBV基因型,了解HBV基因型分布情况。结果 HBV基因分型结果为B型7例(46.7%)、C型4例(26.7%)、B+C型2例(13.3%),另有2例未能分型。结论人群HBV优势基因型以B型为主,C型次之。     

三种食源性致病菌多重PCR检测方法的建立及初步应用

目的建立快速检测沙门菌、变形杆菌和金黄色葡萄球菌的多重聚合酶链反应（PCR）方法。方法本研究依据沙门菌侵袭基因正调节蛋白（hilA）基因、变形杆菌溶血素（hpmA）基因和金黄色葡萄球菌特异性pSa-442序列,运用PrimerPremier5．0分别设计3对特异性引物,预计PCR扩增的目的基因片段分别为为580bp、401bp、256bp。通过对单管多重PCR扩增的特异性、敏感性分析以及建立L16（4^3）正交试验对单管多重PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化,建立了快速同时检测3种食源性致病菌的单管多重PCR方法。结果该方法检测的灵敏度分别为：94．07pg沙门菌DNA,140．85ng变形杆菌DNA,1．41ng金黄色葡萄球菌DNA。模拟检测食品中的混合3种菌,4h培养后样品的最低检测限度分别为：沙门菌10^0菌落形成单位（CFU）／mL、变形杆菌10^1CFU／mL、金黄色葡萄球菌10^0CFU／mL。结论该方法特异性和灵敏度高,检验周期短,可用于对食品中多种致病菌的快速诊检和监控。     

多重荧光错配引物PCR进行单核苷酸多态性检测分析

目的 发展一种快速、检测位点数适中(5～20)、既适用于中小规模科研也适用于临床检测的单核苷酸多态性(SNPs)分型方法.方法 通过改进等位基因特异性聚合酶链反应(PCR),加入公用荧光引物,发展一种利用毛细管电泳的多重荧光错配引物PCR方法,对已知基因型的8份健康者样本的5个线粒体SNPs位点进行分型.结果 一条公用荧光引物即可同时完成5个SNPs位点的分型,其结果与已知基因型完全一致.结论 多重荧光错配引物PCR在中小规模科研及临床检测中具有经济、快速的优势,该方法尤其适用于微量DNA样本的检测.     

一种多重PCR方法快速鉴定7种常见肠道病原菌

目的 建立一种能够快速检测并分型5种致泻性大肠埃希菌、志贺菌及沙门菌的多重聚合酶链反应(multiplex polymerase chain reaction,M-PCR) 方法。 方法 设计针对7种常见肠道致病菌的12对引物,通过多重PCR方法扩增后电泳观察相应条带,确定病原菌。临床标本直接划线接种于肠道选择性平皿,挑取可疑菌落直接提取核酸进行多重PCR检测。 结果 所有标准菌株均能扩增到相应目的片段,322份腹泻病标本中使用该方法共检出志贺菌24株(福氏志贺菌7株、宋内志贺菌17株)、 沙门菌5株、EPEC共12株(均为aEPEC)、ETEC共6株(elt阳性3株;est阳性3株)、EIEC共3株、VTEC共1株(stx1和stx2A均阳性)、EAEC共3株。 结论 该方法快速特异,能同时检测7种常见肠道致病菌,可用于腹泻病常见病原的快速检验。     

A real-time multiplex PCR for the identification and typing of Vibrio cholerae

We report the development and validation of a duo-triplex real-time polymerase chain reaction (PCR) for the rapid identification and typing of Vibrio cholerae. The PCR assay targets a species-specific toxR gene present in all strains of V. cholerae and used as a marker for the species wbeO1 and wbfO139, encoding the O1 and O139 somatic antigens, and ctxA, encoding cholera toxin (CT). The two tcpA variants associated with the classical and El-Tor biotypes are used to infer biotype. The assay was evaluated using 178 isolates comprising eight different Vibrio species, including 122 isolates of V. cholerae. The PCR results of 171/178 (96.1%) isolates were concordant with the serotyping, biotyping, and expected CT results. Variants of toxR (n = 3), nonfunctional wbeO1 (n = 1), and CT-negative isolates of V. cholerae O1 (n = 3) were likely explanations for the mismatched results. This duo-triplex real-time PCR is a reproducible and robust assay for the rapid identification and typing of V. cholerae belonging to the highly pathogenic, pandemic lineages.     

三种食源性致病菌多重PCR检测方法的建立及初步应用

目的建立快速检测沙门菌、变形杆菌和金黄色葡萄球菌的多重聚合酶链反应(PCR)方法。方法本研究依据沙门菌侵袭基因正调节蛋白(hilA)基因、变形杆菌溶血素(hpmA)基因和金黄色葡萄球菌特异性pSa-442序列,运用Primer Premier 5.0分别设计3对特异性引物,预计PCR扩增的目的基因片段分别为为580bp、401 bp、256 bp。通过对单管多重PCR扩增的特异性、敏感性分析以及建立L16(43)正交试验对单管多重PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化,建立了快速同时检测3种食源性致病菌的单管多重PCR方法。结果该方法检测的灵敏度分别为:94.07 pg沙门菌DNA,140.85 ng变形杆菌DNA,1.41 ng金黄色葡萄球菌DNA。模拟检测食品中的混合3种菌,4 h培养后样品的最低检测限度分别为:沙门菌100菌落形成单位(CFU)/mL、变形杆菌101CFU/mL、金黄色葡萄球菌100CFU/mL。结论该方法特异性和灵敏度高,检验周期短,可用于对食品中多种致病菌的快速诊检和监控。     

The role of a real-time PCR technology for rapid detection and identification of bacterial and fungal pathogens in whole-blood samples

The rapid diagnosis of pathogens and prompt initiation of appropriate antibiotic therapy are critical factors to reduce the morbidity and mortality associated with sepsis. In this study, we evaluated a multiplex polymerase chain reaction (PCR-M) test that detects bacteria and fungi in whole-blood specimens, comparing its features to those of a blood culture (BC). Following evaluation of the performance for sensitivity and specificity of PCR-M, 78 blood samples from 54 patients with suspected bacterial infections were evaluated. Whole-blood samples for PCR-M were collected at the same time as BC, and PCR-M results were compared with BC results. As a result, minimum sensitivity of the kit was 1–100 cfu/ml. The PCR-M test correctly identified specificity for 13 out of 14 strains blinded to the assay analyst. Of 78 blood samples examined, 56 (72%) were negative by both methods, and 22 (28%) were positive by at least one of the two methods. PCR-M detected organisms in 21 cases (27%) compared with 12 cases (15%) in BC. The correlation of positives between PCR-M and BC was 92% (11/12), and both methods identified the same organisms in these 11 cases. With higher positive rate compared with BC, PCR-M could detect and identify potentially significant microorganisms within a few hours by using a small volume of a single whole-blood sample. Early detection of microorganisms has the potential to facilitate early determination of appropriate treatment and antimicrobial selection.     

D试验和多重PCR检测红霉素耐药葡萄球菌

目的了解红霉素耐药的葡萄球菌对克林霉素的耐药表型和基因型，指导临床合理使用抗生素。方法用NCCLS标准D试验检测红霉素和克林霉素的耐药表型，通过多重PCR检测耐药基因ermA、ermC和msrA。结果所有144株葡萄球菌中，84株（58．3％）对红霉素和克林霉素耐药（cMLS），27株（18．8％）对红霉素耐药对克林霉素敏感但D试验阳性（iMLS），33株（22．9％）对红霉素耐药，对克林霉素敏感但D试验阴性（MS）。在MS型耐药的菌株中均只检测到msrA基因，在cMLS和iMLS型耐药的菌株中，除了1株3种基因均阴性外，其他均检测到ermA或ermC基因。在金葡菌中iMLS型耐药的菌株以ermC基因稍多（3／5），而cMLS型耐药的菌株以ermA基因居多（56／62）；在凝固酶阴性葡萄球菌中iMLS型和cMLS型耐药的菌株均以ermC基因居多（分别为22／22和11／14）结论iMLS型耐药的葡萄球菌在临床比较多见，多重PCR和D试验均可对其鉴别，临床微生物实验室应常规进行D试验。     

荧光定量PCR检测婴幼儿巨细胞病毒感染情况分析

目的探讨荧光定量PCR(FQ-PCR)方法检测婴幼儿尿液人巨细胞病毒(HCMV)在HCMV感染诊断中的意义。方法采用FQ-PCR检测148例临床疑似HCMV感染婴幼儿尿液中HCMV-DNA水平。同时采用ELISA法检测婴幼儿血清HCMV-IgM抗体。在尿液HCMV-DNA检测阳性患儿中比较血清HCMV-IgM抗体阳性与阴性患儿尿液中HCMV-DNA拷贝数差异。结果 FQ-PCR检测尿液HCMV-DNA的阳性率为35.14%,ELISA检测婴儿血清HCMV-IgM的阳性率为15.54%。两种方法对HCMV感染诊断的阳性率差异有统计学意义(P<0.01)。在尿液HCMV-DNA检测阳性患儿中,IgM抗体阳性组尿液HCMV-DNA拷贝数显著高于阴性组(P<0.01)。结论 FQ-PCR检测尿液HCMV-DNA是早期诊断婴幼儿HCMV感染的敏感有效的方法。     

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection

The development of a quadriplex PCR method with amplification of HCMV in a single‐step procedure using primers taken from four different regions of the viral genome is described. Different concentrations of dNTPs and MgCl₂ were assayed in order to optimize the constitution of the buffer for the multiplex PCR. The specificity of the PCR was tested with 100ng, 10ng, and 1ng of genomic MRC‐5 cell DNA infected with CMV in the presence of 10μg of uninfected MRC‐5 cell DNA. The sensitivity of the PCR was evaluated by the amplification of various amounts (100ng, 10ng, 1ng, and 0.1ng) of genomic MRC‐5 cell DNA infected with CMV. The specificity and sensitivity assays were performed for each pair of primers and for the combined four primer pairs in the multiplex PCR. CMV was consistently detected from 10ng of genomic MRC‐5 cell DNA with each primer pair. When all four sets of primers were combined in a single reaction tube, the sensitivity of the assay was equivalent to 10ng of genomic MRC‐5 cell DNA, whereas amplification from 1ng genomic MRC‐5 cell DNA produced only a subset of the amplimers. By amplifying four target‐sequences of HCMV simultaneously with minimum incubation time at each temperature, a quadriplex, highly sensitive PCR assay was performed. The use of four primer sets designed in different genomic regions of HCMV allowed the detection of variants and achieved maximal sensitivity and specificity which are essential for a diagnostic utilization. J. Clin. Lab. Anal. 13:99–105, 1999. © 1999 Wiley‐Liss, Inc.     

荧光定量聚合酶链反应对生殖器疱疹病毒感染的检测

目的 探讨荧光定量聚合酶链反应（FQ-PCR）在诊断生殖器疱疹病毒感染中的应用。方法 本研究共分两组，检测组即就诊的疑似生殖器疱疹患者347例，对照组12例。分别用FQ-PCR方法检测两组送检标本中的单纯疱疹病毒（HSV）DNA。结果 347例疑似生殖器疱疹患者送检标本中，HSVDNA阳性139例，阳性率为40．1％（139／347），其中阳性标本中有皮损组织液75例、男性尿道拭子31例、女性宫颈分泌物33例，各类标本的阳性率依次为91．5％（75／82）、21．1％（31／147）、28．0％（33／118）。HSVDNA阳性人群中性别差异无统计学意义（P〉O．05，X^2检验）。12例对照标本中没有检出HSVDNA。结论 分泌物中检出的HSVDNA能直观地反映患者当前病毒感染状况，在有皮损组织的患者中，HSVDNA的检出率更高，FQ-PCR能准确、快速地对生殖器疱疹病毒感染做出诊断。     

多重PCR方法检测多耐药鲍曼不动杆菌基因型

目的建立一种适于临床微生物实验室快速检测多耐药鲍曼不动杆菌基因的多重PCR方法。方法筛选临床分离鉴定的多耐药鲍曼不动杆菌105株;用热煮沸法提取DNA,采用多重PCR技术,优化反应条件,同时扩增OXA酶基因(blaOXA-23-like,blaOXA-24-like,blaOXA-51-like,blaOXA-58-like)和整合酶基因(intI1,intI2),扩增产物经凝胶成像系统分析。结果105株多耐药鲍曼不动杆菌中,76株是blaOXA-51-like blaOXA-23-like intI1基因型,18株是blaOXA-51-like intI1基因型,10株是blaOXA-51-like blaOXA-23-like基因型,1株是blaOXA-51-like blaOXA-23-like blaOXA-58-like基因型。未检测出携带有blaOXA-24-like和intI2基因的菌株。结论多耐药鲍曼不动杆菌的耐药性及流行趋势与其携带的OXA酶基因和整合酶基因相关。