Detection of Borrelia burgdorferi DNA by the polymerase chain reaction

DNA amplification by the polymerase chain reaction (PCR) was used to detect DNA of the Lyme disease spirochaete Borrelia burgdorferi. Primers that specify the amplification of a 145 basepair DNA fragment of the OspA gene of B. burgdorferi were used. The amplification product was detected by gel electrophoresis and ethidium bromide staining or by hybridization to a radiolabelled oligonucleotide probe. The hybridization method was found to be more sensitive. As little as 50 fg of purified B. burgdorferi DNA could be detected by PCR. This corresponds to fewer than 50 spirochaetes. The specificity of PCR for B. burgdorferi was tested by using DNA from other organisms as templates for amplification. No cross-reactivity was found. The data shown provide useful information for the development of a PCR-based diagnostic test for Lyme disease.     

Detection of Mycoplasma hyopneumoniae DNA by the polymerase chain reaction

DNA amplification by the polymerase chain reaction (PCR) was examined to detect DNA of Mycoplasma hyopneumoniae, an etiological agent of porcine pneumonia. A pair of synthetic primers was selected that specify the amplification of a 520-basepair DNA fragment in a reiterative sequence of M. hyopneumoniae genome. The PCR product was detected by direct gel electrophoresis or by blot hybridization to a synthetic oligonucleotide probe. The specificity of PCR for M. hyopneumoniae was confirmed by lack of cross-reactivity to DNA from other porcine mycoplasmas.     

Detection of Chlamydia pneumoniae DNA in atheromatous tissues by polymerase chain reaction

Chlamydia pneumoniae is an important human respiratory pathogen. Recently, seroepidemiological data and the demonstration of chlamydial DNA or antigen within parts of atherosclerotic lesions have suggested a causal association between chlamydial infections and atherosclerosis. As the results obtained by different groups show considerable variations, we provide further data based on a highly specific and sensitive nested PCR method. Positivity was confirmed by nonradioactive hybridization with a specific probe, and sensitivity was determined by titration experiments. C. pneumoniae DNA was detected in 8/29 (28%) of carotid artery samples and 3/14 (21%) of aortic aneurysms.     

聚合酶链反应检测解脲脲原体的研究

鉴于临床迫切需要建立特异、敏感、快速的实验诊断方法，从解脲脲原体（ＵＵ）基因组保守区域选择一对寡聚核甘酸引物建立检测ＵＵ的聚合酶链反应（ＰＣＲ）技术，扩增产物片段为１８６ｂｐ，同时采用ＰＣＲ法和培养法对５２份孕妇宫颈分泌物检测。结果ＰＣＲ检出率（７５％）明显高于培养法检出率（２９％），经统计学配对计数资料χ￣２检验有显著性差异（Ｐ＜０．０２５）。研究表明：ＰＣＲ具有特异、敏感、快速等优点，是临床检测ＵＵ切实可行的方法。     

Detection of Histoplasma capsulatum DNA in human samples by real-time polymerase chain reaction

The main aim of our study was to determine the added value of real-time polymerase chain reaction (PCR) for the diagnosis of Histoplasma capsulatum in routine biologic practice. No amplification signal was observed with the 18 non-H. capsulatum strains used to test the specificity of the protocol. The sensitivity threshold of the real-time PCR assay was about 10 fg of H. capsulatum DNA per microliter, tested with a 10-fold serial dilution of the positive control. We analyzed 348 human samples submitted for the routine diagnosis of systemic mycosis. Real-time PCR using the TaqMan system was evaluated against direct microscopic examination and culture. Among the 341 samples without PCR inhibition (n = 7), 66 tested positive by culture, whereas 74 tested positive by real-time PCR. Sensitivity of the real-time PCR assay was estimated at 95.4% and specificity at 96.0% with respect to culture, widely considered to be the gold standard method; however, the molecular approach in fact produced better sensitivity and specificity results. Moreover, for the 38 samples that tested negative by direct examination but positive by culture, the culture method took a mean of 31 days longer than the PCR method to generate results. The protocol presented here may be very useful for improving routine histoplasmosis diagnosis.     

PCR和巢式PCR直接检测血清中HIV核酸序列

采用ＰＣＲ和巢式ＰＣＲ方法对５３例ＨＩＶ抗体阳性和阴性标本进行了检测，以扩增ＨＩＶ特异性ＤＮＡ序列，结果美国ＡｂｂｏｔｔＨＩＶ阳性血清２份，新加坡阳性血清２份，国内阳性血清２份及ＨＩＶ－１病毒培养物１份，两套ＰＣＲ扩增反应物均为阳性，对其中一套ＰＣＲ扩增的ｇａｇ基因序列进行巢式ＰＣＲ也为阳性。采自西南边境地区的２３份ＥＬＩＳＡ和ＷＢ阳性血清，各种ＰＣＲ反应均为阳性；１１份ＥＬＩＳＡ可疑阳性、ＷＢ阴性标本中，有两例血清呈ＰＣＲ阳性，探针杂交亦为阳性。而１２例ＳＩＶ、ＳＲＶ及猴血清标本ＰＣＲ反应均为阴性。上述结果表明，采用ＰＣＲ方法检测血清中ＨＩＶ核酸序列是一种很有前途的ＨＩＶ感染诊断方法。     

套式PCR法检测外周血肺炎衣原体DNA

目的探讨外周血单个核细胞(PBMC)中肺炎衣原体(Cpn)DNA的检测方法.方法用淋巴细胞分层法和低渗溶血法提取PBMC.设计2对引物用套式PCR(nPCR)法测定150例冠心病患者(冠心病组)外周血PBMC中Cpn-DNA.同期临床55例非冠心病患者作对照组.结果冠心病患者肺炎衣原体DNA阳性率32.7%,对照组阳性率为1.8%,两组差异显著(P＜0.001),OR 26.2,95%CI 3.52～194.98.冠心病组中无症状组阳性率为29.1%,不稳定型心绞痛组阳性率为39.6%,急性心肌梗死组阳性率为29.9%,各亚组间差异不显著(P＞0.05).结论用nPCR法检测PBMC中Cpn-DNA,可为临床Cpn感染的诊断提供依据.     

Detection of hepatitis B virus DNA using the polymerase chain reaction technique

The polymerase chain reaction (PCR) technique has been utilized for the detection of hepatitis B virus (HBV) DNA, and several factors related to the selection of primer pairs for the PCR amplification have been demonstrated. The sensitivity of the PCR assay was compared with that of slot-blot hybridization for detecting HBV-DNA. Analysis by the PCR technique with Southern blot hybridization provided a greater than 10(4)-fold increase in sensitivity over the slot-blot hybridization analysis. Also, a rapid and sensitive PCR method for the detection of serum HBV-DNA was developed: HBV-DNA is released from virions by incubating serum with NaOH followed by neutralization with HCl. HBV-DNA sequences are then detected by agarose gel electrophoresis and ethidium bromide staining after PCR amplification with successive sets of primer pairs. In testing serial samples from chimpanzees experimentally infected with HBV, HBV-DNA was detected 2-3 wk before the appearance of hepatitis surface antigen (HBsAg) and continued to be detectable for a short period after the production of antibody to HBsAg. Results from testing of human serum demonstrated that the majority of patients with HBsAg in serum had HBV-DNA as well and that some patients had HBV-DNA in serum in the absence of HBsAg.     

Differentiation of mycobacteria in sputa by duplex polymerase chain reaction for mycobacterial hsp65 gene

Early differentiation of mycobacteria in sputa is crucial. This study was set to evaluate the usefulness of a newly developed duplex polymerase chain reaction (PCR) for hsp65 gene-based method in differentiating mycobacteria in sputum with a positive acid-fast bacilli (AFB) smear before culturing. One hundred forty-seven sputa with positive AFB smear were included for the analysis. Mycobacterial species were identified using a newly developed duplex PCR for hsp65 gene followed by a nested PCR-direct sequencing and the conventional colony-based method. Final decision of mycobacterial species were made based on 1) results of species identification based on mycobacterial colonies or 2) results of species identification of other sputa from the same patients and clinical findings. The duplex PCR-based method correctly identified 83.2% sputa from tuberculosis patients and 82.2% sputa from nontuberculous mycobacteria patients, whereas the colony-based method correctly identified 86.1% and 77.8%, respectively. Sensitivity and specificity of the colony-based method for Mycobacterium tuberculosis were 86.1% and 100%, respectively, whereas those of the duplex PCR-based method were 83.2% and 95.6%, respectively. The duplex PCR-based method, to differentiate mycobacterial species in sputa, produced comparable results as those of the colony-based identification method.     

Detection of toxin-producing pathogenic bacterial strains by polymerase chain reaction

Polymerase chain reaction (PCR) was used for detection of pathogenic Clostridium botulinum, Clostridium perfringens, Clostridium difficile, and Escherichia coli. With this aim in view, primers to botulinic toxins types A, B, C1, D, E, F, and G, perfringens enterotoxin, difficile toxin, and types 1 and 2 Shigella-like toxins were chosen and synthesized. Optimal amplification conditions were selected for each pair of primers, with DNA and the respective agent as the reaction mixture matrices. PCR was highly specific and sensitive in all cases. Its sensitivity was 10-100 cells/sample. Among the tested C. botulinum, C. perfringens, C. difficile, and E. coli strains, specific amplification products of expected size were observed only in the strains containing the respective toxin genes. These findings recommend the use of these methods in clinical microbiology. Strains containing type 2 Shigella-like toxin gene were detected among E. coli strains isolated from patients with the hemolytic uremic syndrome, which for the first time indicates that the problem with E. coli epidemic strain O157 is valid for Russia. As a result of our studies, test systems for detection of types A, B, C, D, E, F, and G C. botulinum strains, C. perfringens and C. difficile, and E. coli O157 strains are now available.     

Detection of microsatellite instability by fluorescence multiplex polymerase chain reaction

We have created a clinical molecular diagnostic assay to test for microsatellite instability (MSI) at multiple loci simultaneously in paraffin-embedded surgical pathology colon resection specimens. This fluorescent multiplex polymerase chain reaction (PCR) assay analyzes the five primary microsatellite loci recommended at the 1997 National Cancer Institute-sponsored conference on MSI for the identification of MSI or replication errors in colorectal cancer: Bat-25, Bat-26, D2S123, D5S346, and D17S250. Amplicon detection is accomplished by capillary electrophoresis using the ABI 310 Genetic Analyzer. Assay validation compared 18 specimens previously assessed by radioactive PCR and polyacrylamide gel electrophoresis detection to results generated by the reported assay. Germline and tumor DNA samples were amplified in separate multiplex PCR reactions, sized in separate capillary electrophoresis runs, and compared directly to identify novel length alleles in tumor tissue. A concordance of 100% between the two modalities was achieved. The multiplex assay routinely detected a subpopulation of 10% tumor alleles in the presence of 90% normal alleles. A novel statistical model was generated that corroborates the validity of using results generated by analysis of five independent microsatellites to achieve a single overall MSI diagnosis. The assay presented is superior to standard radioactive monoplex PCR, polyacrylamide gel electrophoretic analysis, primarily due to the multiplex PCR format.     

Detection of cytomegalovirus in blood donors by the polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed and optimized to detect cytomegalovirus (CMV) DNA in the blood of 86 normal donors who had originally tested seropositive for CMV. Evidence of previous or current infection with CMV was determined by rescreening of the blood for CMV antibodies and by detecting the presence of infectious virus in the white cells by cell culture. DNA was extracted from the blood of donors by a manual or an automated method and amplified by PCR using primers from the major immediate early gene of CMV DNA. The amplified product was detected by visualization of a fluorescent 435-base pair DNA band in an electrophoretic agarose gel after ethidium bromide staining and confirmed by slot-blot DNA hybridization using an oligonucleotide probe with complementarity for the major immediate early gene. Seven (8%) of the 86 donors were positive for CMV DNA in both fluorescence and hybridization studies. These donors were also antibody positive. While 74 (86%) of the 86 donors were positive for the presence of CMV antibodies in enzyme-linked immunosorbent assay, none was positive for virus in cell culture. PCR has the potential to be an effective and reliable procedure for the detection of CMV DNA in donor blood, but further study is required for this technique to be used for diagnostic or routine screening purposes.     

乙型肝炎病毒DNA荧光定量PCR的建立与应用

目的　建立血浆 (血清 )HBVDNA荧光定量PCR检测方法 ,探讨其临床应用价值。方法　用PUCm T载体和PCR纯化产物连接 ,转染DH5α菌 ,筛选阳性菌落 ,提取质粒 ,制作外标准品 ;在HBV基因组S区设计一对引物和TaqMan探针 ,严格优化反应物的组成和扩增条件 ,并对该方法进行评价。结果　建立的HBVDNA荧光定量PCR最低可检出 80 0拷贝 /ml;批内误差为 1 8%～ 6 5% ,批间误差为 2 6 %～ 9 1 % ;在 1 0 4 ～ 1 0 11拷贝 /ml之间与Ct值具有很好的线性 (Ct =- 1 .391 1ln(X) 4 1 .6 5,r =- 0 .9958) ;外周血HBVDNA临床定量检测发现 ,HBeAg阳性的 1 30例中 ,HBVDNA阳性率为 1 0 0 % ,含量为1 0 6 89± 1.6 9拷贝 /ml,抗HBe阳性的 96例中HBVDNA阳性率为 55 2 % ( 53/ 96 ) ,含量为 1 0 4 .0 9± 1.19拷贝 /ml;6 2例献血员未检出HBVDNA。乙肝临床治疗监测发现 ,外周血中HBVDNA含量检测可有效反映治疗效果 ,指导临床用药。结论　荧光定量PCR检测方法是一种相对准确、灵敏度高、特异性较强和较简便的HBVDNA定量检测技术 ,对乙肝诊断、指导临床用药和治疗监测方面具有实用价值     

聚合酶链反应检测肺炎衣原体方法的建立与初步应用

目的探讨聚合酶链反应（ＰＣＲ）技术在肺炎衣原体检测中的应用。方法参照Ｃａｍｐｂｅｌ报道的肺炎衣原体全基因序列、Ｇ＋Ｃ比例和次级结构设计一对引物，采用ＰＣＲ法检测肺炎衣原体ＤＮＡ及临床标本中的肺炎衣原体感染。结果经对肺炎衣原体、沙眼衣原体、大肠埃希菌、溶血性链球菌、白色念珠菌和结核分枝杆菌培养物进行ＰＣＲ扩增，结果只有肺炎衣原体出现单一４３７ｂｐ的扩增区带；敏感性试验可检测出１０ｆｇ的肺炎衣原体ＤＮＡ；１０份模拟标本均扩增出４３７ｂｐ区带；２２份小儿肺部感染的咽拭子标本中有５份为阳性，阳性率为２２．７％，而显微镜检查只有２例可见包涵体；１２份正常小儿咽拭子标本均为阴性。结论ＰＣＲ法检测肺炎衣原体，具有特异、敏感、快速、准确的特点，为肺炎衣原体感染的临床诊断提供了科学的依据     

Detection of Chlamydia trachomatis by use of polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. One primer set was used, from the published sequence of the common C. trachomatis plasmid. Detection of amplified sequences was carried out by agarose gel electrophoresis. Analysis of 106 clinical samples tested by cell culture and PCR showed a sensitivity of 100% when PCR was compared with cell culture.     

Detection of parvovirus B19 by dot-blot and polymerase chain reaction

Our studies compare detection of parvovirus B19 DNA by dot-blot hybridization and by the polymerase chain reaction (PCR). Compared to detection by dot-blot hybridization with a 32P-oligonucleotide probe, the sensitivity of PCR product detection by ethidium bromide fluorescence is not compromised when the size of the amplification target and number of cycles of amplification are properly selected. Our PCR assay simultaneously amplifies two targets in the B19 genome and yields a sensitivity 10 times greater than 32P-labelled oligonucleotide--dot-blot hybridization (dot-blot). In 126 serum samples from cases of suspected parvovirus B19 infection, viral DNA was detected by PCR in 17% (21/126), whereas dot-blot detected B19 DNA in 0.8% (1/126). Parvovirus B19-specific antibodies were detected in 69% (87/126) of the suspected clinical cases by immunoblot. Nineteen of the antibody-positive specimens were DNA positive of which three were IgM-positive/IgG-negative, 11 IgM-positive/IgG-positive and five IgM-negative/IgG-positive. The only dot-blot DNA positive sample was also PCR-DNA positive but was B19 IgM-negative/IgG-negative. Eighty-six B19 antibody-negative, clinically uninfected controls were also negative for B19 DNA by PCR and by dot-blot. These studies confirm the increased sensitivity of gene amplification by PCR for detection of B19 DNA and demonstrate that two targets in B19 DNA can be amplified simultaneously with resultant increased ease of interpretation. Finally, detection of the two B19-specific products by ethidium bromide fluorescence is documented.     

聚合酶链反应检测呼吸道感染非细菌性多种病原体的研究

目的　评估聚合酶链反应 (PCR)方法在呼吸道感染病原学诊断方面的应用价值。方法　呼吸道感染患者 (感染组 )和健康人 (对照组 )组成研究对象 ,采集呼吸道排泌物 (鼻咽分泌物和痰 )为检测标本 ,应用聚合酶链反应(PCR)方法检测腺病毒 (ADV)、巨细胞病毒 (CMV)、柯萨奇病毒 (COX)、呼吸道合胞病毒 (RSV)、肺炎衣原体 (CP)、沙眼衣原体 (CT)、肺炎支原体 (MP)和解脲支原体 (UU)。结果　感染组的阳性检测结果分别为 :ADV 11.5 %、CMV14 .5 %、COX 18.0 %、RSV 2 9.5 %、CP 34.5 %、CT 4 .5 %、MP 17.0 %、UU 5 .5 % ;除CT和UU外 ,其他病原体的结果与对照组比较 ,差异均具有统计学意义 (P <0 .0 1)。结论　PCR方法具有高特异性和敏感性 ,可以作为呼吸道感染病原学诊断方法应用 ;CP、RSV、COX、MP等微生物可能是导致呼吸道感染重要的病原体。     

Detection of enterotoxigenic Escherichia coli in stool specimens by polymerase chain reaction

A polymerase chain reaction (PCR) protocol for rapid (7 h) detection of enterotoxigenic Escherichia coli (ETEC) is described. This protocol has been validated on 57 stool samples from young children by comparing it with the colony hybridization technique. A good agreement was found between the two methods with Cohen’s kappa statistics of 0.87 and 0.79 for the detection of the heat-stable toxin (ST) and heat-labile toxin (LT), respectively. Of 26 samples positive for LT and 15 samples positive for ST by colony hybridization, 21 (81%) and 15 (100%) were also found to be positive for LT and ST by PCR, respectively. Only one sample identified as LT-negative by colony hybridization was found to be positive by PCR. However, 3 of 42 samples of ST-negative by colony hybridization were detected as positive by PCR. A reconstruction experiment revealed that PCR could detect LT-producing and ST-producing ETEC at minimal concentrations of 2.5 × 10³ cfu and 2.5 × 10² cfu per gram of feces, respectively. These data indicate the possible use of this method for rapid identification of ETEC-associated diarrhea in clinical and epidemiological settings.     

聚合酶链反应检测克隆性免疫球蛋白重链基因重排

目的建立较简便、敏感的Ｂ细胞恶性肿瘤微小残留病（ＭＲＤ）检测方法。方法根据免疫球蛋白重链（ＩｇＨ）基因的保守序列设计引物，应用半巢式聚合酶链反应（ＰＣＲ）基因扩增技术检测克隆性ＩｇＨ基因重排。结果该方法敏感性达１０－３。６２例Ｂ细胞恶性肿瘤患者，５１例检测到克隆性ＩｇＨ基因重排，阳性率为８２％，假阴性率为１８％（１１／６２）。同时检测３５例非Ｂ细胞恶性肿瘤患者和２０例正常人均阴性，无假阳性。检测１４例Ｂ细胞非何杰金淋巴瘤患者形态学检查正常的骨髓标本，ＭＲＤ检出率为７１％（１０／１４）。随访５例处于完全缓解期的Ｂ细胞型急性淋巴细胞白血病患者，６个月内ＭＲＤ均阳性，１２个月后３例转阴性，２例持续阳性，１８个月后持续阳性的２例患者均复发，而转阴性的３例仍处于完全缓解状态。结论半巢式ＰＣＲ检测克隆性ＩｇＨ基因重排，方法简便，敏感性和特异性高，可用于Ｂ细胞恶性肿瘤ＭＲＤ检测