肺炎衣原体套式聚合酶链式反应检测研究

目的 探讨肺炎衣原体（Ｃｐｎ）的套式聚合酶链式反应（ｎＰＣＲ）检测技术。方法 根据Ｃａｍｐｂｅｌｌ克隆的ＣｐｎＤＮＡ序列设计套式引物，并建立ｎＰＣＲ反应体系。结果 Ｃｐｎ经ｎＰＣＲ扩增出现３７８ｂｐ的阳性条带，而沙眼衣原体和鹦鹉热衣原体以及其他用作特异性试验的微生物均不能扩增。３例阳性标本经ＤＮＡ测序与Ｃｐｎ（ＣＷＬ－２９株）序列完全一致。Ｃｐｎ ｎＰＣＲ之灵敏度比传统ＰＣＲ高１００倍。各种呼吸系     

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。     

地高辛素标记弓形虫DNA探针的制备及在产前诊断的应用

本文建立了地高辛素标记的弓形虫ＤＮＡ探针并在２３例产前咨询孕妇中作产前诊断。通过聚合酶链反应扩增弓形虫ＲＨ株ＴＧＲ４核酸片段，扩增产物７７８ｂｐ片段经寡核苷酸层析柱纯化后，用地高辛素标记作为探针，与待检材料斑点杂交。结果显示该探针与待杂交的弓形虫ＲＨ、ＺＳ１、ＺＳ２、ＳＨ１株ＤＮＡ杂交；而与恶性疟原虫、卡氏肺孢子虫、杜氏利什曼原虫、大肠杆菌、巨细胞病毒ＤＮＡ无杂交。并能在待杂交液中检测出相当于１ｐｇ水平ＤＮＡ，表明该探针具有高度的特异性及敏感性。检测２３例孕期１０—１４周孕妇中，阳性３例，其中一孕妇外周血、绒毛组织及羊水均呈阳性。另一例的外周血及绒毛组织呈阳性。该探针稳定、敏感、操作简单，具有推广意义。     

16S—23S rDNA间隔区序列PCR扩增及探针杂交用于偶然分支杆？…

目的 从基因水平上确认福建省南平市注射后偶然分支杆菌暴发感染的致病菌,并建立ＰＣＲ结合ＤＮＡ探针杂交快速鉴定偶然分支杆菌的方案。方法 根据偶然分支杆菌１６Ｓ－２３Ｓ ｒＤＮＡ间隔区序列,设计合成一段寡核苷酸探针,采用ＰＣＲ扩增、ＲＦＬＰ和ＤＮＡ斑点杂交技术,对２９例偶然分支杆菌临床分离株进行鉴定。结果 ２９例偶然分支杆菌临床分离株均被ＰＣＲ扩增出一条约４７０ｂｐ的ＤＮＡ条带,ＤＮＡ探针杂交也出现特     

聚合酶链反应快速检出耐甲氧西林凝固酶阴性葡萄球菌的研究

目的快速检出耐甲氧西林凝固酶阴性葡萄球菌。方法利用聚合酶链反应（ＰＣＲ）检测耐甲氧西林凝固酶阴性葡萄球菌，建立一种从葡萄球菌中快速提取ＤＮＡ方法，用粗提物为模板，检测编码耐甲氧西林葡萄球菌青霉素结合蛋白的ｍｅｃＡ基因，扩增产物为５３３ｂｐ大小。结果药敏法鉴定为耐甲氧西林凝固酶阴性葡萄球菌２１株，１００％ｍｅｃＡ基因阳性，２３株对甲氧西林敏感的凝固酶阴性葡萄球菌中有９株用ＰＣＲ扩增出ｍｅｃＡ基因片段。利用ｒ－３２Ｐ标记寡核苷酸作为探针，Ｓｏｕｔｈ－ｅｒｎ杂交结果与ＰＣＲ检测结果完全一致，证实５３３ｂｐ片段为耐甲氧西林凝固酶阴性葡萄球菌ｍｅｃＡ基因特异性片段。结论ＰＣＲ方法具有快速、敏感的优点，并能特异地检出临界（ＭＩＣ＝０．５～２ｍｇ／Ｌ）耐甲氧西林凝固酶阴性葡萄球菌株。     

PCR 与 DIFA 检测沙眼衣原体的比较研究

用所建立的沙眼衣原体质粒引物聚合酶链反应技术（ＰＣＲ）检测１１６例泌尿生殖道沙眼衣原体，并与直接免疫荧光法（ＤＩＦＡ）进行比较。结果ＰＣＲ阳性３８例，ＤＩＦＡ阳性的３２例中有３１例ＰＣＲ阳性。ＰＣＲ敏感性为９６．９％，特异性为９１．７％。ＰＣＲ阳性者于治疗结束１～２周后复诊２１例，其中４例ＰＣＲ仍然阳性。结果显示，ＰＣＲ可快速、敏感、特异地检测泌尿生殖道沙眼衣原体ＤＮＡ，然而对治疗后短期内复诊患者，似乎不宜仅以ＰＣＲ阳性结果作为重复治疗的依据。     

16s～23s rDNA间隔区序列聚合酶链反应鉴定术后龟分支杆菌暴发感染

目的　从基因水平上确认深圳市某医院术后暴发感染的致病菌,并建立一套快速的ＰＣＲ方法鉴定龟分支杆菌脓肿亚种。方法　根据分支杆菌的１６ｓ～２３ｓｒＤＮＡ间隔区序列,设计合成一对引物和龟分支杆菌脓肿亚种ＤＮＡ探针,采用ＰＣＲ 和ＤＮＡ 斑点杂交技术,对５３ 株龟分支杆菌脓肿亚种临床分离株进行ＰＣＲ扩增和ＤＮＡ杂交。在此基础上,采用１６ｓ～２３ｓｒＤＮＡＰＣＲ扩增体系检测２５９份临床标本,对部分标本做ＤＮＡ探针斑点杂交反应。结果　５３ 株龟分支杆菌脓肿亚种临床分离株均被扩增出一条特异的３８０ ｂｐ ＤＮＡ 带和出现特异的杂交斑点。２５９ 份临床标本,ＰＣＲ 扩增阳性率为６０－６％ 。结论　在基因水平上确认此次引起院内术后暴发感染的致病菌为龟分支杆菌脓肿亚种。１６ｓ～２３ｓｒＤＮＡＰＣＲ扩增检测体系灵敏、特异,能鉴定龟分支杆菌。     

mip基因聚合酶链反应结合地高辛标记探针杂交鉴定嗜肺军团菌

目的对嗜肺军团菌国外参考菌株及国内分离菌株的巨噬细胞感染增强子（ｍｉｐ）基因分析，建立一种快速、敏感、特异的嗜肺军团菌基因检测鉴定方法。方法利用嗜肺军团菌种特异性ＤＮＡ片段ｍｉｐ基因通过聚合酶链反应（ＰＣＲ）扩增，对ＰＣＲ阳性产物进行地高辛标记的ｍｉｐ基因探针杂交，观察其反应结果。并对２６株环境和患者中分离的疑似嗜肺军团菌菌株进行分析。结果所有实验用嗜肺军团菌国外参考菌株及国内分离菌株的ｍｉｐ基因的ＰＣＲ扩增结果都为阳性，探针杂交结果也为阳性，而非嗜肺军团菌及与非军团菌对照菌株的ＰＣＲ扩增结果及探针杂交结果都为阴性，本实验其灵敏度和特异度均为１００％。另外２６株疑似嗜肺军团菌菌株有６株被鉴定为嗜肺军团菌。结论所建立嗜肺军团菌的快速初步鉴定方法具有很高的特异性和敏感性，并具有较高的应用价值     

PCR检测老年呼吸道疾病患者肺炎衣原体感染临床研究

肺炎衣原体 (ｃｈｌａｍｙｄｉａｐｎｅｕｍｏｎｉａＣＰｎ)又称ＴＷＡＲ组衣原体 ,1989年定名为衣原体属内的第 3种 ,在世界上多个国家和地区均有流行。主要引起人的非典型肺炎、支气管炎等呼吸系统疾病。由于肺炎衣原体只能在活细胞内生长繁殖 ,因此 ,用细胞培养法分离检测肺炎衣原体时间长 ,方法复杂 ,分离率低。而血清学检测只能做回顾性诊断 ,使肺炎衣原体的临床诊断受到限制。本文用快速、敏感、特异的聚合酶链式反应 (ＰＣＲ)技术对广州地区部分老年呼吸疾病患者进行了肺炎衣原体特异性ＤＮＡ检测 ,以了解老年呼吸疾病患者肺炎衣原体…     

PCR结合非放射性标记DNA探针技术用于班氏微丝蚴检测的研究

本研究根据班氏丝虫ＩＷｂ３５重复序列，用聚合酶链反应扩增、回收班氏丝虫特异性片段（６５６ｂｐ），以异羟基洋地黄毒苷配基（Ｄｉｇｏｘｉｇｅｎｉｎ，Ｄｉｇ）标记该片段，制备非放射性ＤＮＡ探针，并把ＰＣＲ和Ｄｉｇ标记ＤＮＡ探针杂交检测方法结合起来，用于检测安徽班氏丝虫病流行区病人血样中的微丝蚴。结果表明，该探针与马来丝虫ＤＮＡ及人白细胞ＤＮＡ不发生杂交反应，仅与班氏微丝蚴ＤＮＡ发生杂交反应，可检出０．５ｐｇ同源ＤＮＡ。微丝蚴血样经ＰＣＲ扩增后与探针进行斑点杂交试验，能检出６０μｌ血样中１条微丝蚴，３１例流行区班氏微丝蚴阳性患者，检出阳性３０例，３２例流行区“正常人”检出阳性２例，３２例非流行区正常人均阴性。     

肺炎衣原体包涵体核酸组分衍化分析法的建立

吖啶橙（ＡＯ）染色可以作为肺炎衣原体（Ｃｐｎ）包涵体的染色方法。它是一种快速检查Ｃｐｎ感染的较好方法，有可能替代荧光单抗染色的功能，克服单抗染色时细胞结构显示不清的缺点，具有简捷、价廉和既有反映衣原体包涵体的形态和成份，又能反映包涵体不同时期ＲＮＡ和ＤＮＡ含量变化的独特优点。     

聚合酶链反应检测弓形虫的实验研究

目的建立敏感、特异、稳定的聚合酶链反应（ＰＣＲ）法,以检测弓形虫感染。方法建立ＰＣＲ法,确定最佳扩增条件;用ＰＣＲ法检测实验感染家兔血液中ＤＮＡ的动态变化,并与ＥＬＩＳＡ法检测ＣＡｇ进行比较。结果兔感染弓形虫后第２ｄ,ＰＣＲ开始出现阳性,阳性率为７６．９％（１０／１３）,总检出率达１００％。与ＥＬＩＳＡ法查ＣＡｇ进行平行检测,前者出现阳性时间早,且阳性率明显高于后者。该法敏感性高,可检测到１０ｆｇＤＮＡ含量;特异性强,对其它９种寄生虫和微生物ＤＮＡ均无交叉现象;稳定性好,对阳、阴性同一样品重复５次,结果一致。结论提示本法可用于临床诊断,在一定范围内替代病原学和免疫学检测。     

儿童上呼吸道感染者的生殖支原体套式PCR检测与DNA测序研究

目的　生殖支原体（Ｍｇ）是从人体分离到的第１２ 种支原体,并认为与男女性泌尿生殖道的炎性相关。１９８８ 年,国外学者曾从４ 例肺炎患者的咽部分离到Ｍｇ。我们检测了２０ 例正常儿童和６０ 例上呼吸道感染儿童的咽试子标本中的Ｍｇ１６ ＳｒＲＮＡ基因。目的在于探讨Ｍｇ 与儿童上呼吸道感染的关系。方法　本文采用灵敏、特异的套式ＰＣＲ检测技术,并对１例阳性标本和Ｍｇ（Ｇ－３７Ｔ）典型株作了ＤＮＡ测序分析。结果　６０ 例儿童上呼吸道感染者中有１３ 例Ｍｇ 阳性,其中１ 例经ＤＮＡ测序与Ｍｇ（Ｇ－３７Ｔ）典型株完全一致。结论　Ｍｇ 可能是儿童上呼吸道感染的病因之一     

PCR快速检测致病性钩端螺旋体DNA

以四株不同型别的致病性钩端螺旋体ＤＮＡ为模板进行聚合酶链反应（ＰＣＲ），建立了钩体快速检测方法。在此基础上，先后对流行期１５例早期疑似血清进行ＰＣＲ快速检测，结果７例阳性，与经防疫部门及临床确诊的７例患者完全符合，阳性率为１００％。而常规方法（显微镜凝集试验）只检出５例阳性。结果表明，ＰＣＲ用于钩体病的早期论断，快速、准确。     

以地高辛标记探针研究人16型乳头瘤病毒感染与良性前列腺增生症的相关性

目的用分子生物学方法制备地高辛标记１６型人乳头瘤病毒（ＨＰＶ１６）ＤＮＡ探针，对ＨＰＶ与良性前列腺增生症（ＢＰＨ）的相关性进行探讨。方法将质粒ｐＨＰＶ１６经ＢａｍＨＩ酶切，Ｖ－形槽电洗脱回收ＨＰＶ１６ＤＮＡ，随机引物法进行标记。用斑点杂交对探针的质量参数进行评估，并对２９例ＢＰＨ和７例正常前列腺组织进行了ＨＰＶ１６ＤＮＡ检测。结果表明本探针灵敏特异、经济实用。ＢＰＨ组和正常对照组的阳性率分别为６２．１％和１４．３％，两者差异有显著性（Ｐ＜０．０５）。结论ＢＰＨ可能是ＨＰＶ感染的又一男性宿主，ＨＰＶ１６感染可能在ＢＰＨ中起着一定的病因学作用。     

Detection of Chlamydia pneumoniae by polymerase chain reaction-enzyme immunoassay in intestinal mucosal biopsies from patients with inflammatory bowel disease and controls

BACKGROUND AND AIM: It has been suggested that Chlamydia is an organism that may have the potential to cause inflammatory bowel disease (IBD) in susceptible individuals. Chlamydia pneumoniae has emerged as an important human pathogen in the last decade. The objective of the present study was to investigate the frequency of the presence of C. pneumoniae DNA in intestinal biopsies from patients with IBD and from non-IBD controls. METHODS: The DNA was extracted from 222 colonoscopic biopsies, which were obtained from 11 patients with Crohn's disease (CD), 18 patients with ulcerative colitis (UC) and from 37 non-IBD control patients. The presence of the C. pneumoniae omp1 gene and C. trachomatis 16S rRNA gene was determined using a rapid and sensitive polymerase chain reaction-enzyme immunoassay (PCR-EIA). RESULTS: The C. pneumoniae-specific DNA was detected in 32 (14.4%) of 222 endoscopic biopsies. Among them, C. pneumoniae DNA were found in nine of 42 (21.4%) biopsies from patients with CD, nine of 59 (15.3%) biopsies from patients with UC, and 14 out of 122 (11.4%) biopsies from non-IBD control patients, respectively. Moreover, the percentage of patients with at least one biopsy positive for C. pneumoniae was higher, although not statistically significant, in CD (36.4%) and UC patients (38.9%) compared to non-IBD controls (16.2%). In contrast, C. trachomatis DNA was detected in only two of 222 (0.9%) biopsy samples. CONCLUSION: The C. pneumoniae DNA was detected in the intestine of both patients with IBD and in non-IBD control patients, probably reflecting the high prevalence of this organism in the environment. The moderate yield of positive biopsies in our IBD patients and the fact that the detection rate of C. pneumoniae DNA was similar in endoscopic biopsies from IBD patients and non-IBD controls does not support a direct role for this organism in the pathogenesis of IBD.     

The role of atypical respiratory pathogens in exacerbations of chronic obstructive pulmonary disease.

The aetiology of acute exacerbations of chronic obstructive pulmonary disease (COPD) is heterogeneous and still under discussion. Serological studies have suggested that Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila may play a role in acute exacerbations of COPD. The presence of these atypical pathogens in sputum samples was investigated in patients with stable COPD and with acute exacerbations of COPD using real-time PCR. The present study was part of a randomised, double-blind, single-centre study and a total of 248 sputum samples from 104 COPD patients were included. In total, 122 samples obtained during stable disease (stable-state sputa) and 126 samples obtained during acute exacerbations of COPD (exacerbation sputa) were tested. Of the 122 stable-state sputa, all samples were negative for M. pneumoniae and C. pneumoniae DNA, whereas one sample was positive for Legionella non-pneumophila DNA. Of the 126 exacerbation sputa, all samples were negative for M. pneumoniae and C. pneumoniae DNA, whereas one sample was positive for Legionella non-pneumophila DNA. The possible relationship between the presence of atypical pathogens and the aetiology of acute exacerbations in chronic obstructive pulmonary disease was investigated in patients with stable disease and in those with acute exacerbations using real-time PCR. No indication was found of a role for Legionella spp., Chlamydia pneumoniae or Mycoplasma pneumoniae in stable, moderately severe chronic obstructive pulmonary disease and in its exacerbations.     

应用聚合酶链反应快速检测临床标本中的肺炎支原体

应用聚合酶链反应（ＰＣＲ）技术检测临床标本中的肺炎支原体（ＭＰ）。在１４０例非细菌性肺炎患者的标本中，ＰＣＲ试验阳性３０例。其中间接血凝试验阴性面ＰＣＲ试验阳性者２１例。ＰＣＲ阳性标本经ＭＰ５－４寡核苷酸探针检测验证，均为阳性结果。     

Acute respiratory Chlamydia pneumoniae infections in adults. Value of direct gene amplification diagnosis

Chlamydia pneumoniae has been established recently as an important human respiratory pathogen. The aim of this study was to define the incidence of Chlamydia pneumoniae in acute respiratory infections by evaluating its presence in posterior nasopharyngeal aspirates or broncho-alveolar lavage specimens by polymerase chain reaction-hybridization (PCR-EIA) as well as the titres of specific antibodies in serum by a rELISA test and a micro-immunofluorescence (MIF) test. 68 adults patients were investigated. Eight patients (11.8%) were positive by either rELISA or PCR-EIA or both, with an infection rate of 5 patients with community-acquired pneumonia, 2 asthmatic patients and 1 patients with an exacerbation of chronic obstructive pulmonary disease. Serological evidence of acute infection was found in four of these patients with rELISA test and in three others with MIF test. PCR-EIA detected Chlamydia pneumoniae DNA in four patients, but there were concordant results with rELISA and PCR-EIA in only one patient. In conclusion, Chlamydia pneumoniae appears to be a common etiologic agent of acute respiratory infections in adults. The discrepancy between serological test and PCR-EIA results reflects the difficulties in routine laboratory diagnosis of Chlamydia pneumoniae. The ambiguous results of serological tests from a single serum sample assess the utility of PCR for prompt diagnosis. When PCR is negative or no feasible, a second serology to 15/21 days of interval is necessary. Further studies with optimised techniques must be developed.     

鹦鹉热衣原体套式PCR和DNA测序方法研究

目的　建立特异、灵敏、快速的鹦鹉热衣原体 (Cps)分子生物学检测方法。 方法　采用套式PCR扩增检测和DNA测序方法 ,并进行实验室评价。结果　 4株Cps均能被套式PCR扩增出 2 14bp目的条带 ,而沙眼衣原体、肺炎衣原体、肺炎支原体等其它微生物均不能检出 ;Cps套式PCR灵敏度高于CpsPCR ;模拟样本均能被检出。Cps(CS株 )套式PCR产物直接测序与Cps典型株序列完全一致。结论　套式PCR是检测Cps特异、灵敏 ,且快速的分子生物学检测方法