胃粘膜石蜡切片PCR法检测幽门螺杆菌

目的建立从石蜡包埋的胃粘膜标本中扩增ＨｐＤＮＡ的聚合酶链反应（ＰＣＲ）．方法以一对Ｈｐ特异的寡核苷酸为引物，采用双扩增ＰＣＲ扩增Ｈｐ１６ＳｒＲＮＡ基因，并与常规检测方法进行了比较．结果双扩增ＰＣＲ检出的最小ＨｐＤＮＡ量为００１ｐｇ．用该方法检测十二指肠溃疡患者２０例石蜡包埋的胃粘膜标本，并与尿素酶试验、细菌培养、Ｗａｒｔｈｉｎ＿Ｓｔａｒｒｙ银染色及ＰＣＲ对新鲜胃粘膜标本的检测作比较，结果１８例患者上述五项检测结果一致，２例尿毒酶试验、银染色及ＰＣＲ对新鲜胃粘膜标本的检测呈阳性的患者，采用双扩增ＰＣＲ对石蜡包埋标本的检测未发现ＨｐＤＮＡ．ＰＣＲ对石蜡包埋标本及新鲜胃粘膜标本的检测有显著相关性．结论双扩增ＰＣＲ为分子水平上Ｈｐ感染的回顾性研究提供一有利工具．     

聚合酶链反应和DNA探针联合检测临床标本中结核杆菌的研究

目的为提高聚合酶链反应（ＰＣＲ）检测未培养临床标本中结核杆菌的敏感性和特异性，方法，首先通过琼脂糖凝胶电泳检测ＰＣＲ扩增产物，然后用Ｓｏｕｔｈｅｒｎ法转移到至膜上，与地高辛标记的人型结核杆菌ＤＮＡ探针杂交。结果ＰＣＲ电泳检测的灵敏度为１ｐｇ而ＤＮＡ探针检测将灵敏度提高到１００ｆｇ。２４种受试菌株中，只有结核分支杆菌复合体和蟾分杆菌有２４５ｂｐ扩增带，其中牛型结核分支杆菌与１８８ｂｐ探针不杂交，对２     

套式聚合酶链式反应诊断恶性疟原虫及混合感染的研究

为了建立一种特异、敏感、简便的疟疾诊断方法,设计并合成两对针对恶性疟原虫（Ｐ．ｆ．）小亚单位核糖体核糖核酸（ＳＳＵｒＲＮＡ）基因特异的引物,采用套式聚合酶链式反应（ＰＣＲ）技术,扩增恶性疟原虫ＳＳＵｒＲＮＡ基因特定片段。利用该系统诊断云南及海南疟疾患者血样,并随机选取扩增产物进行克隆测序鉴定。结果显示,恶性疟原虫模板均扩增出长度为２０５ｂｐ的特定基因片段,经Ｔ－载体克隆测序与恶性疟原虫ＳＳＵｒＲＮＡ特定基因片段序列完全符合。该系统特异性强,除恶性疟原虫外,间日疟原虫（Ｐ．ｖ．）、三日疟原虫（Ｐ．ｍ．）、卵形疟原虫（Ｐ．ｏ．）、正常人血ＤＮＡ样本及空白对照均无此扩增带出现。该系统检测原虫的敏感度为１．５个原虫／μｌ,大大高于常规镜检。６１份Ｐ．ｆ．镜检阳性标本在进行ＰＣＲ检测时亦均呈阳性,同时联合应用套式ＰＣＲ扩增间日疟原虫ＳＳＵｒＲＮＡ特定基因片段的检测系统,发现６１例恶性疟病人中２３例是Ｐ．ｆ．和Ｐ．ｖ．混合感染。该检测系统具有特异、敏感、稳定、简便的特点,对疟疾的诊断、混合感染的判定具有一定的临床应用价值     

PCR法检测胃粘膜中的幽门螺杆菌

目的建立检测幽门螺杆菌（Ｈｐ）的更为敏感的聚合酶链反应（ＰＣＲ），并探讨它在Ｈｐ感染的诊断及根除效果判定中的应用．方法以一对合成的与Ｈｐ１６ＳｒＲＮＡ基因互补的寡核苷酸为引物（ＣＰ１／ＣＰ２），建立了从胃粘膜中检测Ｈｐ的ＰＣＲ反应，并与常规检测方法进行比较．结果ＰＣＲ方法检测Ｈｐ标准菌株及５０株临床分离菌株均产生５００ｂｐ片段，检出的最小ＤＮＡ量为０１ｐｇ，相当于１００个细菌细胞；所有１３株其它细菌及无Ｈｐ感染的人胃粘膜则无扩增产物出现．用该方法检测９６名初诊患者Ｈｐ感染情况及２１名药物治疗后患者Ｈｐ根除情况，并与胃粘膜活组织尿素酶试验、细菌培养及银染色方法比较，证实ＰＣＲ方法可以检出常规方法不能检出的少量Ｈｐ．结论ＰＣＲ是检测Ｈｐ的最为敏感的方法，有助于Ｈｐ感染的诊断和Ｈｐ根除的精确判断     

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。     

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。     

多聚酶链反应快速检测衣原体

应用沙眼衣原体内源性质粒引物和鹦鹉热衣原体１６ｓｒＲＮＡ基因引物，对沙眼衣原体眼部感染和尿道感染进行快速检测，４９份临床诊断为沙眼的标本经ＰＣＲ检测，阳性３１份，阴性１８份；２８份临床诊断为非淋菌性尿道炎的标本，经ＰＣＲ检测，阳性１１份；阴性１７份。以上结果分别与国际公认的免疫荧光试剂盒和酶联免疫试剂盒对同一标本的检测结果进行了比较，表明多聚酶链反应技术远较ＩＦＡ和ＥＬＩＳＡ灵敏，是快速检测沙眼衣     

肺癌炎性疾病者生殖支原体套式PCR检测研究

探讨自生殖支原体与肺部炎性疾病的关系,方法应用原核生物通用引物ＰＣＲ直接测序克隆Ｍｇ１６ＳｒＲＮＡ基因片段序列,自行建立Ｍｇ套式ＰＣＲ扩增法,检测健康儿童和肺部炎性疾病患儿咽拭子标本中的Ｍｇ－ＤＮＡ。结论Ｍｇ可能与肺癌炎性疾病相关联。     

rDNA探针杂效检测病人痰标本中结核杆菌的研究

目的 应用结核分枝杆菌特异的ｒＤＮＡ探针杂交检测痰标本中的结核杆菌ｒＤＮＡ,评价其在临床标本检测中的应用价值。方法 用引物ｂ对结核分枝杆菌１６Ｓ－２３ＳｒＤＮＡ间隔区序列进行扩增,同时加入生物素标记制成２５０ｂｐ的ｒＤＮＡ探针。对该探针的敏感性、特异性进行了研究,并对９０分结核病人,３０份非结核病人痰标本的ＰＣＲ产物进行了斑点杂交检测。结果 ｒＤＮＡ探针检测引物ｂ增产物杂交只有结核分枝杆菌、胃分枝     

疟原虫DNA模板制备用于PCR诊断方法的研究

为确立一种快速，敏感，特异的疟原虫检测方法，根据红内期疟原虫ＳＳＵｒＲＮＡ基因序列，设计合成引物３条，采用微量全血和滤纸干血滴标本快速制备疟原虫ＤＮＡ模板的９种方法，进行聚合酶链反应（ＰＣＲ）并比较。结果显示有５种方法的实验效果最佳，其中，全血ＰＣＲ仪预热及滤氏干血滴Ｃｈｅｌｅｘ煮沸两法操作简单，所需试剂较少。采用该两法对深圳地区２０２份和湖北地区１６份镜检阳性的全血和深圳地区１２９份滤纸干血滴标     

Use of polymerase chain reaction (PCR) and DNA probe hybridization to determine the Gram reaction of the infecting bacterium in the intraocular fluids of patients with endophthalmitis

OBJECTIVES: To evaluate polymerase chain reaction (PCR) combined with DNA probe hybridization to determine the Gram reaction of the bacterium in intraocular specimens from patients with infectious endophthalmitis. METHODS: Fifty-seven intraocular specimens - 17 aqueous humor (AH) and 40 vitreous fluid (VF) - from 55 patients with clinically diagnosed infectious endophthalmitis and 25 control intraocular specimens from non-infectious ocular disorders (10 AH and 15 VF) were evaluated by microscopy, culture and PCR-DNA probe hybridization to detect the Gram reaction of the bacterium. RESULTS: PCR-DNA probe hybridization was specific and sensitive to detect 30 fg of both gram-positive and gram-negative bacterial DNA. None of the controls showed bacteria by microscopy, culture or PCR. Of the 57 intraocular specimens, conventional microbiological methods could detect a bacterial aetiology in 32 (56.1%), while PCR-DNA probe hybridization could detect 52 (91.2%) specimens. This difference was statistically significant (P= 0.003). In bacteriologically positive specimens, there was absolute correlation of the Gram reaction between the results of smear and culture methods and PCR-DNA probe hybridization. Of the 25 bacteriologically negative specimens, 20 (80%) were positive by PCR-DNA probe hybridization, of which seven (35%) were gram-positive, 12 (60%) gram-negative and one (5%) positive by both. Results of PCR on AH and VF were not significantly different. CONCLUSION: PCR and DNA probe hybridization to determine the Gram reaction of the bacterium in intraocular fluids is a specific and sensitive method in the diagnosis of bacterial endophthalmitis. AH is an ideal specimen for PCR, since its collection is a simple and safe office procedure.     

16S rRNA基因芯片诊断新生儿败血症

目的建立16SrRNA基因加基因芯片检测新生儿败血症的诊断技术，以提高临床检测细菌的速度及准确性。方法对125例拟诊为败血症的新生儿血标本及部分脑脊液标本进行细菌16SrRNA基因及基因芯片检测．包括DNA提取、设计引物和探针、聚合酶链反应（PCR）扩增、基因芯片的制备、杂交、激光扫描与读片。结果125例中PCR检测血标本阳性64例，占51．2％；血培养阳性32例，占25．6％；非特异性指标41例，占32．8％，差异有统计学意义（P〈0．01）。若以血培养阳性和／或非特异指标至少两项阳性为诊断标准，PCR灵敏度为90．3％（56／62），特异度为87．3％（55／63），正确诊断指数为0．776。3例脑脊液标本中PCR阳性2例，培养阳性仅1例。对64例PCR阳性血标本进一步作基因芯片检测，结果通用探针均阳性。其中G^＋探针阳性60份。G探针阳性4份。30份PCR和血培养均阳性的标本中其探针菌株与血培养细菌阳性结果相符。2例脑脊液标本PCR阳性，基因芯片检测结果与培养结果相符。结论168 rRNA基因PCR加基因芯片杂交可为新生儿败血症提供早期、敏感的病原学诊断依据。     

Simple microplate hybridization assay for detection of amplified products of Mycobacterium tuberculosis

We describe a simple microplate hybridization assay for the rapid detection of the IS6110 PCR products of Mycobacterium tuberculosis from clinical cultures and from sputum specimens. The assay is based on the specific detection with a fluorescein-labeled detection probe of biotinylated PCR products which are captured on avidin coated microplate. Hybridized products with fluorescein were identified by using anti-fluorescein antibody, horseradish peroxidase conjugate and colorimetric peroxidase substrate. The specificity of the assay was assessed by analysis of 56 bacterial strains: the assay discriminated perfectly between the positive and negative groups when an OD490 of 0.18 was used as the cut-off point. The assay was sensitive enough to detect as little as 1 pg of M. tuberculosis H37Rv DNA, which is equivalent to approximately three bacilli. To evaluate the assay performance clinically, 190 sputum samples from newly diagnosed TB patients were tested; 79 were classified as TB positive, and 111 were classified as TB negative by culture and acid-fast staining as the 'gold standard'. The sensitivity, specificity and accuracy of the PCR-microplate hybridization assay were 90, 100 and 96%, respectively. The total assay time of hybridization following the PCR was 4 hours. The PCR-microplate hybridization assay is fast, simple and accurate and is suitable for use in the microbiology laboratory or for the analysis of large numbers of samples.     

Diagnostic application of a polymerase chain reaction assay for the Whipple's disease bacterium to intestinal biopsies

BACKGROUND & AIMS: The uncultured Whipple's disease bacterium (Tropheryma whippelii) was characterized in 1991-1992 by polymerase chain reaction (PCR) and sequencing of the bacterial 16S ribosomal RNA gene. The aim of this study was to develop a PCR assay for diagnostic purposes. METHODS: Modified primers for PCR and a specific probe for hybridization were designed. The specificity of this PCR assay was tested using 37 bacterial control strains and intestinal biopsy samples from 16 patients without Whipple's disease. The sensitivity was tested in 88 intestinal biopsy samples from 35 patients with Whipple's disease. RESULTS: PCR and hybridization were negative in all 37 bacterial controls and in all 16 patients without Whipple's disease. Before therapy, DNA of T. whippelii was detected in all 30 patients with Whipple's disease from whom formalin-fixed biopsy material was available, whereas Bouin-fixed material was negative. During and after treatment, PCR was negative in 23 of the 24 patients who were followed up. Generally, conversion to negative occurred within 1 year. Despite negative intestinal PCR, symptomatic cerebral Whipple's disease appeared in 3 patients. CONCLUSIONS: This PCR assay is specific and sensitive and is applicable as a diagnostic test. However, PCR from intestinal biopsy samples seems less helpful for monitoring the effect of treatment. (Gastroenterology 1996 Jun;110(6):1735-43)     

Enhanced Detection of HIV-1 Sequences Using Polymerase Chain Reaction and a Liquid Hybridization Technique

In this report we describe a sensitive HIV-1 detection method which is applicable for confirmation testing of donors whose blood sample gives indeterminate viral-serology results. The method involves performing a polymerase chain reaction (PCR) and detecting the generated fragments using liquid hybridization and gel retardation. We found that it is as specific as blotting on a filter and hybridization with an internal probe but at least tenfold more sensitive. After applying it on DNA samples of a panel of 11 persistent indeterminate anti-p24gag-reactive donors, none was found to be PCR positive. Considering other negative virological and biochemical test results and case-historical data, these donors are not likely to be HIV-1 infected.     

Detection of human cytomegalovirus in clinical specimens by DNA-DNA hybridization

A diagnostic assay has been developed to detect human cytomegalovirus (HCMV) DNA in clinical specimens with 32P-labeled cloned fragments of HCMV strain AD169. The labeled probe can detect 10 pg of HCMV DNA and fails to hybridize to DNA from other herpesviruses or human DNA. The assay correctly identified 22 (92%) of 24 coded urine specimens culture positive for HCMV and 23 (88%) of 26 urine specimens culture negative for HCMV. In a prospective study of 67 buffy-coat specimens from recipients of bone marrow transplants HCMV DNA was detected in 13 (93%) of 14 culture-positive samples. Of 53 buffy-coat specimens culture negative for HCMV, 32 were hybridization negative for HCMV DNA. However, in 20 of 21 buffy-coat specimens positive for HCMV by hybridization but negative by culture there was evidence that the hybridization assay was correct and more sensitive than currently available tissue culture techniques.     

常见致病菌23S rRNA基因片段序列分析及其应用初探

目的通过临床常见致病菌23S rRNA基因序列差异的分析，探索建立可同时检测或鉴定多种细菌的分子生物学方法。方法对常见致病菌23S rRNA基因的一个多变区序列进行聚合酶链反应（PCR）扩增和测序分析，根据序列差异设计相应引物和探针，并采用PCR-凝胶电泳分析和PCR-反向杂交技术检测或鉴定临床常见致病菌。结果1.革兰阳性菌和革兰阴性菌间的23S rRNA基因序列存在较大差异，通过PCR扩增和凝胶电泳分析能快速区分待测菌株为革兰阳性菌抑或革兰阴性菌。2.不同菌种间亦存在一定差异，可通过PCR-反向杂交技术将细菌鉴定到种。结论.临床常见致病菌的23S rRNA基因之间存在可供细菌鉴定的序列差异，据此可望建立各种具有快速，灵敏，准确特点的分子生物学方法，为细菌感染的快速病原诊断提供客观依据。     

Development of a duplex-polymerase chain reaction for rapid detection of pathogenic Leptospira

A duplex-polymerase chain reaction (PCR) for the rapid detection of pathogenic leptospires was developed by using two sets of newly designed primers which amplified in the same reaction two different DNA fragments simultaneously: 279-bp of LipL32 and 430-bp of 16S rRNA. For DNA extraction from bacterial cultures, the silica-based spin column method was found to be more suitable and was selected for the extraction of DNAs from all 92 bacterial strains including 56 strains of pathogenic Leptospira, 15 strains of non-pathogenic Leptospira and 21 other strains of bacteria. The PCR products were analyzed by agarose gel-electrophoresis with confirmation by Southern and dot hybridization using synthetic DNA probe prepared from LipL32 gene of a pathogenic reference strain, L. interrogans serovar pyrogenes. The duplex-PCR allowed detection of two products of 279 bp and 430 bp in all pathogenic Leptospira. Non-pathogenic Leptospira generated a single product of 430 bp. Other bacterial strains failed to reveal any amplification products. As little as 1 pg of pure DNA corresponding to 100 cells could be detected by agarose gel-electrophoresis, and 1-10 fg of pure DNA by hybridization.     

Comparison of gram stain, DNA probe, and culture for the identification of species of Mobiluncus in female genital specimens

The detection of species of Mobiluncus in female genital specimens by DNA probe (with whole-chromosomal bacterial DNA), culture, and gram stain were compared by using specimens obtained from hospital patients, college students, and women attending a sexually transmitted disease clinic. Culture purification and speciation required an average of 37 days to complete, whereas the DNA-probe assay required five days. Gram stain was also rapid but did not allow speciation. There was 100% correlation between species identification by DNA probe and by conventional biochemical tests. Gram stain alone detected 90% of the samples that were positive by any method, whereas culture detected 77%-83% and DNA probe detected 52%-83%.     

对硝基苯甲酸生长试验鉴别结核与非结核分枝杆菌应用评价

目的对应用对硝基苯甲酸（PNB）生长试验鉴别结核分枝杆菌复合群（MTC）与非结核分枝杆菌（NTM）进行评价。方法采用PNB生长试验、PCR-反向核酸探针杂交和PCR-DNA直接测序对52株MTC和264株NTM临床分离株进行鉴定。结果以PCR-反向核酸探针杂交和PCR-DNA测序结果为鉴定标准,PNB生长试验中52株MTC菌株均为（100％）阴性（-）,264株NTM菌株中233株（88％）为阳性（＋）,31株（12％）为（-）。结论仅应用PNB生长试验鉴别MTC与NTM存在局限性。用PCR-反向核酸探针杂交能将分枝杆菌鉴定至属、将MTC与NTM相鉴别,并能将NTM准确鉴定至种的水平。