聚合酶链反应检测淋病奈瑟菌

聚合酶链反应检测淋病奈瑟菌殷常红，倪语星，王祥兴淋病奈瑟菌（ＮＧ）是性传染疾病的主要病原之一。传统的涂片染色和分离培养法其敏感性和特异性还不能达到临床的要求。本文作者将ＰＣＲ技术用于检测ＮＧ，旨在为临床上诊断淋病提供新的敏感、特异和快速的方法。材料...     

脑脊液细菌16S rRNA荧光定量聚合酶链反应快速诊断儿童化脓性脑膜炎

临床确诊化脓性脑膜炎（化脑）主要依赖于脑脊液（CSF）细菌培养，但该法耗时较长，阳性率不高，而且确诊前常因有不正规的抗生素治疗使CSF改变更不典型等，给早期诊治带来一定的困难，难以适应临床需要。为快速、可靠地诊断化脑，我们对20例本院疑似化脑患儿进行CSF细菌DNA荧光定量测定，监测其CSF细菌DNA拷贝数、CT值，同期与CSF细菌培养的对照，效果较好，现报道如下。     

聚合酶链反应快速诊断结核性脑膜炎

聚合酶链反应快速诊断结核性脑膜炎唐小平，罗惠娟，黄已实，卢群馨近年来，国外陆续报道用聚合酶链反应（ＰＣＲ）诊断结核性脑膜炎（结脑），显示出独特的优点。我们应用ＰＣＲ技术检测了５０例结脑患者的脑脊液（ＣＳＦ）标本，并与其它方法的检测结果进行比较，现将结...     

聚合酶链反应快速诊断流行性脑脊髓膜炎

采用聚合酶链反应（ＰＣＲ）扩增脑膜炎奈瑟氏菌（Ｎｍ）ＩＳ１１０６插入序列的方法诊断流行性脑脊髓膜炎（流脑）。所试２１株Ｎｍ均扩增产生预期长度５９６ｂｐ的特异性片段，１４株非Ｎｍ菌株未见此片段。扩增灵敏度为１２ｆｇ。Ｎｍ菌ＩＳ１１０６重复序列扩增产物构成特征性ＰＣＲ指纹图，Ａ群菌株图谱一致；Ｂ群菌株呈明显多态性变化。临床标本的ＰＣＲ检测结果为：２１份流脑患者脑脊液中２０份为阳性（９５．２％）；１４份流脑患者急性期血清中１２份阳性（８５．７％）；在所检标本中Ｎｍ培养阳性的８份脑脊液和４份急性期血清ＰＣＲ检查也同样阳性。作为对照的１２份恢复期血清、３份密切接触者血清和２０份正常人血清均为阴性。作者认为，ＩＳ１１０６－ＰＣＲ是诊断流脑的一种简便快速、灵敏特异的方法。     

连接酶链反应(LCR)技术检测男性尿标本的淋病奈瑟菌

目的应用连接酶链反应（LCR）技术检测男性尿标本中的淋病奈瑟菌,初步评价其敏感性和特异性。方法采集受检者晨起或较长时间（2小时以上）不排尿后的首段尿（FVU）标本1131例,利用LCR检测此尿液标本中的淋球菌,针对Cut-off值在灰区以上的标本进行聚合酶链反应（PCR）检测。对LCR和PCR结果相异的标本,用另一LCR试剂复检,参照“扩大的金标准”来确定检测结果。结果LCR的敏感性和特异性分别为100％和99．9％。结论LCR作为一种探针检测技术,是一种既敏感又特异的诊断方法,可避免取尿道标本给患者带来的痛苦,可作为筛检男性淋病奈瑟菌感染的一种非损伤性方法。     

PCR法检测16S rRNA基因在败血症诊断中的价值

目的评价外周血细菌16S rRNA基因检测诊断败血症的敏感性及特异性。方法利用通用引物对6种常见细菌16S rRNA基因进行扩增，通过阳性菌株及阴性对照检测引物的特异性；观察不同退火温度及不同细菌浓度下PCR检测效果；检测败血症患者及正常人血液标本中的16S rRNA基因表达，并与血培养结果进行比较，评价其诊断意义。结果6种阳性菌株均出现特异阳性条带，阴性质控未出现阳性条带；不同退火温度对PCR结果无明显影响，以55、58℃最佳；PCR方法的最低细菌检测浓度为1．5×10^3cfu／ml；观察组血培养阳性率为38．3％（23／60），血液16S rRNA基因阳性表达率为86．6％（52／60），P〈0．01。结论PCR法检测16S rRNA基因用于败血症诊断特异性及敏感性强，但应注意规范操作、避免污染。     

应用聚合酶链反应检测病原性耶尔森氏菌

目的 用聚合酶链反应检测病原性Yersinia(耶尔森氏菌)属菌。方法 通过病原性Yersinia属菌3对引物，进行基因扩增，并且用凝胶电泳分离，照像。结果 病原性Yersinia属菌得到目的扩增带，非病原性Yersinia属菌及其他细菌没有扩增带。结论 聚合酶链反应方法简便，易行，特异性强，敏感性高。     

内蒙古部分炭疽杆菌16S和23S rRNA基因间段分析

为了解在内蒙古分离的炭疽杆菌与其它地区的菌株之间有无差异,采用ＰＣＲ加酶切的方法对拨和的被试菌进行了１６Ｓ和２３Ｓ ｒＲＮＡ基因间段分析。结果发现所有实验菌株间的ＰＣＲ及酶切图谱一致,说明我区的炭疽杆菌与其它地区的菌株在遗传学上具有同源性,无本质差异。     

聚合酶链反应检测肝硬化患者腹水中细菌DNA

目的:探讨PCR方法检测肝硬化患者腹水中细菌DNA的可行性.方法:在细菌16S rRNA基因保守区设计一对通用引物,对7种对照菌株、人基因组DNA、HBV DNA、37份肝硬化患者腹水进行聚合酶链反应扩增.结果:7种对照菌株均获得530 bp DNA片段.而与人基因组DNA、HBV DNA无交叉阳性反应,敏感性试验可检测出1pg的细菌DNA.37份腹水中有9份获得53bp DNA片段,阳性率24.3%(9/37),而腹水细菌培养阳性率为5.4%(2/37),两者比较差异有显著性意义(P＜0.05).结论:将通用引物通过PCR方法扩增细菌16S rRNA基因,具有高度敏感性、特异性,可应用于肝硬化患者腹水中细菌DNA的检测及细菌移位的研究.     

快速检测细菌并革兰分型的半巢式聚合酶链反应及其初步应用

目的以半巢式聚合酶链反应(PCR)检测细菌及作革兰阴、阳性分型,并与细菌培养法比较。方法以细菌16SrRNA基因为靶序列,采用一对通用引物(pm1,pm3)和一条革兰阴性型特异性引物(pm2),以半巢式PCR方法扩增实验室保留菌株的DNA并作出革兰染色分型;以人类外周血白细胞基因组DNA、HBVDNA阳性血清以及白假丝酵母菌为对照,检测此方法的特异性;采用倍比稀释菌液作敏感性实验;与细菌培养法比较,验证此方法检测临床标本的敏感性。结果对17个实验室保留菌株进行检测,以通用引物对作第1次PCR均得到371bp长度的DNA片段;再以革兰阴性菌特异引物对(pm2,pm3)作第2次PCR,9种阴性菌均得到353bp的DNA片段,而8种阳性菌未被扩增。特异性实验表明,此通用引物与人类基因组DNA、真菌及病毒无交叉反应。敏感性实验表明,采用半巢式PCR可检测出3个CFU的细菌。对120份临床标本检测,半巢式PCR检测阳性率(29.2%)显著高于细菌培养法检测阳性率(17.5%)。结论此半巢式PCR检测细菌方法,具有特异、敏感、快速的特点,并能对细菌进行革兰阴性、阳性分型,可用于临床感染性疾病的初步诊断。     

实时荧光定量PCR检测直肠癌XPC基因表达

目的 检测核苷酸切除修复（NER）基因XPC在直肠癌及直肠组织中的表达。方法采用实时定量荧光PCR法,检测16例手术后切除的新鲜直肠癌组织及6例癌旁直肠组织中XPC基因表达水平。结果直肠癌中XPC基因水平明显高于正常直肠组织。结论高表达的XPC基因直接在NER早期发挥着重要作用,在一定程度上导致直肠癌患者对化疗药物的敏感性降低。     

16SrRNA基因PCR加反相杂交技术检测细菌DNA

目的探讨聚合酶链反应（ＰＣＲ）加反相杂交技术在细菌ＤＮＡ检测中的应用。方法以１６ＳｒＲＮＡ基因为靶序列，设计引物及寡核苷酸探针，采用ＰＣＲ法加反相杂交检测标准菌株及临床标本细菌ＤＮＡ。结果对２０株不同标准菌株进行ＰＣＲ扩增，均出现３７１ｂｐ长度的ＤＮＡ片段，敏感性试验可检测出１０－１２ｇ的细菌ＤＮＡ，与人基因组及病毒无交叉反应；２２份血培养阳性标本及４份脑脊液培养阳性标本均扩增出３７１ｂｐ长度ＤＮＡ条带，反相杂交区分革兰阳性／阴性细菌与培养结果相符。结论１６ＳｒＲＮＡ基因ＰＣＲ加反相杂交技术检测细菌ＤＮＡ，具有敏感、快速、准确的特点，为细菌感染的临床诊断提供了科学的依据。     

Diarrhoea-predominant irritable bowel syndrome distinguishable by 16S rRNA gene phylotype quantification

AIM: To study whether selected bacterial 16S ribosomal RNA (rRNA) gene phylotypes are capable of distinguishing irritable bowel syndrome (IBS).METHODS: The faecal microbiota of twenty volunteers with IBS, subdivided into eight diarrhoea-predominant (IBS-D), eight constipation-predominant (IBS-C) and four mixed symptom-subtype (IBS-M) IBS patients, and fifteen control subjects, were analysed at three timepoints with a set of fourteen quantitative real-time polymerase chain reaction assays. All assays targeted 16S rRNA gene phylotypes putatively associated with IBS, based on 16S rRNA gene library sequence analysis. The target phylotypes were affiliated with Actinobacteria, Bacteroidetes and Firmicutes. Eight of the target phylotypes had less than 95% similarity to cultured bacterial species according to their 16S rRNA gene sequence. The data analyses were made with repeatedmeasures ANCOVA-type modelling of the data and principle omponent analysis (PCA) with linear mixed-effects models applied to the principal component scores. RESULTS: Bacterial phylotypes Clostridium cocleatum 88%, Clostridium thermosuccinogenes 85%, Coprobacillus catenaformis 91%, Ruminococcus bromii-like, Ruminococcus torques 91%, and R. torques 93% were detected from all samples analysed. A multivariate analysis of the relative quantities of all 14 bacterial 16S rRNA gene phylotypes suggested that the intestinal microbiota of the IBS-D patients differed from other sample groups. The PCA on the first principal component (PC1), explaining 30.36% of the observed variation in the IBS-D patient group, was significantly altered from all other sample groups (IBS-D vs control, P = 0.01; IBS-D vs IBS-M, P = 0.00; IBS-D vs IBS-C, P = 0.05). Significant differences were also observed in the levels of distinct phylotypes using relative values in proportion to the total amount of bacteria. A phylotype with 85% similarity to C. thermosuccinogenes was quantified in significantly different quantities among the IBS-D and control subjects (-4.08 ± 0.90 vs -3.33 ± 1.16, P = 0.04) and IBS-D and IBS-M subjects (-4.08 ± 0.90 vs -3.08 ± 1.38, P = 0.05). Furthermore, a phylotype with 94% similarity to R. torques was more prevalent in IBS-D patients' intestinal microbiota than in that of control subjects (-2.43 ± 1.49 vs -4.02 ± 1.63, P = 0.01). A phylotype with 93% similarity to R. torques was associated with control samples when compared with IBS-M (-2.41 ± 0.53 vs -2.92 ± 0.56, P = 0.00). Additionally, a R. bromii-like phylotype was associated with IBS-C patients in comparison to control subjects (-1.61 ± 1.83 vs -3.69 ± 2.42, P = 0.01). All of the above mentioned phylotype specific alterations were independent of the effect of time.CONCLUSION: Significant phylotype level alterations in the intestinal microbiotas of IBS patients were observed, further emphasizing the possible contribution of the gastrointestinal microbiota in IBS.     

Detection of immunoglobulin gene rearrangement in acute and chronic lymphoid leukemias of B-cell lineage by polymerase chain reaction gene amplification

Polymerase chain reaction (PCR) was used to amplify the DNA fragments of the complementarity determining region 3 of the immunoglobulin (Ig) gene heavy chain from the leukemic cell specimens of patients with acute and chronic lymphoid leukemias of B-cell lineage. Two different pairs of primers were tested. Fourteen of the 17 (82%) cases of acute lymphoblastic leukemia (ALL), and all 15 cases (100%) of B-cell chronic lymphocytic leukemia, who had rearrangement of the Ig gene heavy chain by Southern analysis, were positive by PCR with either one or both pairs of primers. This technique was able to detect leukemic cells at the level of 0.1%. Applying it to study the remission marrow specimens following induction chemotherapy was more useful than morphology alone in predicting early relapse of the leukemia.     

实时荧光定量PCR检测五日热巴通体

目的 采用TaqMan-MGB探针建立检测五日热巴通体的实时荧光定量PCR（real-time quantitative PCR）方法。方法 根据五日热巴通体特异的16-23S rRNA间隔区序列设计引物和探针，以克隆的16-23S rRNA间隔区基因片段作DNA模板，建立了检测五日热巴通体实时荧光定量检测方法。结果 建立的定量标准曲线的循环阈值（Ct）与模板拷贝数呈良好的线性关系（r=0．996）；与普通PCR相比较，荧光定量PCR检测的灵敏度约为它的200倍。用荧光定量PCR检测其它相关立克次体和细菌，检出结果为阴性。实时荧光定量PCR检测五日热巴通体实验感染小鼠血、脾脏、肝脏和肺组织DNA样本，脾脏和肝脏样本中检出较高水平五日热巴通体DNA，血和肺部检出的目的DNA的水平较低。结论 本研究建立的检测五日热巴通体的实时荧光定量PCR法具有很高的检测灵敏度和特异性，可用于临床患者血样本的检测，作五日热的早期诊断。     

Determination of mutations of the 23S rRNA gene of Helicobacter pylori by allele specific primer-polymerase chain reaction method

BACKGROUND AND AIMS: Susceptibility to clarithromycin of Helicobacter pylori (H. pylori) is caused by single nucleotide polymorphisms (SNPs) of the 23SrRNA gene. Allele specific primer-polymerase chain reaction (ASP-PCR) is one of the methods for determining SNPs, which can measure SNPs easily within a short period by PCR amplification alone without digestion with restriction enzymes. The aim of the present study was to develop the ASP-PCR assay for determining SNPs at positions 2,142 and 2,143 of the 23S rRNA gene of H. pylori. METHODS: In total, 112 patients with H. pylori infection based on positive results of a rapid urease test (RUT) were enrolled in the study. Thirty-five had failed to eradicate H. pylori by a clarithromycin-based regimen. DNA was extracted from the RUT-positive gastric tissue samples. SNPs from adenine to guanine at positions 2,142 and 2,143 of the 23S rRNA of H. pylori (A2,142G and A2,143G) were determined by the ASP-PCR method. Minimum inhibitory concentrations (MICs) of clarithromycin for H. pylori were also measured. RESULTS: Forty-nine of 112 patients were infected with wild-type strains of H. pylori. Thirty-nine patients were infected with strains with A2,143G mutations. Twenty-three patients were infected with both wild-type strains and those with A2,143G mutations. Only one patient was infected with the strain with A2,142G mutation. H. pylori strains with A2,143G or A2,142G mutation had significantly higher MICs for clarithromycin. CONCLUSION: The ASP-PCR assay for 23S rRNA mutation of H. pylori is a useful method to detect clarithromycin-resistant strains of H. pylori easily.     

Cardiobacterium valvarum临床分离株的生物学特性研究及16S rRNA鉴定

目的 采用不同的方-法描述Cardiobacterium valvarum(C. valvarum)临床分离株的生物学特性,利用16S rRNA基因测序技术进行分子鉴定。 方-法 转种阳性血培养标本到血琼脂平板上进行细菌培育,革兰染色涂片镜检,用VITEK 2 Compact全自动微生物鉴定分析仪对临床分离株进行细菌鉴定,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)对分离株的蛋白质进行高通量测定,E-test法对分离株作药敏试验。提取分离株的DNA,采用16S rRNA基因测序技术对PCR的产物进行测序,在NCBI的BLAST网站上与GenBank数据库上的序列做相似性比较,用MEGA7.0.26软件构建该分离株的系统进化树。结果 经细菌培养发现小而圆、光滑、不透明,灰色的菌落;经革兰染色后镜下见到小、两端圆形、革兰阴性的多形性杆菌;VITEK 2 Compact上机、MALDI-TOF-MS技术均未得到该分离株的鉴定结-果;16S rRNA基因测序技术测得该菌株基因全序列约为1 450 bp,与C. valvarum F0432的16S rRNA同源性为99.59%,鉴定为C. valvarum。结论该菌的形态、生化反应均无代表性,采用16S rRNA基因测序技术可以对C. valvarum进行鉴定,对该疾病的诊断具有重要意义。     

肠球菌为主要菌群的腹泻标本的16S rRNA与PFGE分析

目的了解肠球菌为主要菌群的腹泻标本的微生态及分离肠球菌菌株的克隆特征。方法根据细菌16S rRNA保守区序列设计通用引物,以腹泻病人粪便标本中的总DNA为模板,PCR扩增16S rRNA片段。将获得的片段克隆入T载体进行测序,与数据库中发表的序列进行比对,判断标本中细菌的种类和比例,并对每份标本分离的各50株肠球菌选择20株进行PFGE分析。结果对4份标本的多次16S rRNA序列分析表明,该4份标本均以Enterococcus faecium为主(所占比例>68%),PFGE结果显示每个病人分离屎肠球菌菌株是克隆性的(一份标本除外)。结论从16S rRNA和PFGE的角度对腹泻粪便标本进行了分析,得到腹泻儿童肠球菌分布的基本特征,其致病性和相关机理还待今后实验范围的进一步扩大和研究的深入。     

聚合酶链反应快速检出耐甲氧西林金黄色葡萄球菌的研究

目的 建立耐甲氧西林金黄色葡萄球菌的快速检出方法。方法 利用聚合酶锭反应（ＰＣＲ）快速检出耐甲氧西林金黄色葡萄球菌（金葡菌），建立一种从葡萄球菌中快速提取ＤＮＡ方法，以粗提ＤＮＡ作为ＰＣＲ模板，检测编码耐甲氧西林金葡菌青霉素结合蛋白２（ＰＢＰ２ａ）的ｍｅｃＡ基因。结果 １８４金葡菌有ＰＣＲ方法及药敏法比较，药敏法鉴定为耐甲氧西林金葡菌（ＭＲＳＡ）５８株，仅一株ＰＣＲ扩增ｍｅｃＡ基因阴性，１２６株甲