二步温控法聚合酶链反应检测脑脊液结核杆菌DNA

应用二步温控法聚合酶链反应（ＰＣＲ）检测脑脊液结核杆菌微量ＤＮＡ，并扩增出标准人型结核杆菌１５８ｂｐ的基因片段。实验结果表明：ＰＣＲ用于结核性脑膜炎患者脑脊液结核杆菌ＤＮＡ的检测，与传统检验方法相比较，具有敏感、快速、特异、高效等优点，是基因水平上检测结核杆菌的一项新技术，用于结核性脑膜炎的早期诊断，具有很强的实用价值，值得临床推广应用。     

聚合酶链反应-微孔板杂交技术用于结核分枝杆菌的检测

目的建立结核分枝杆菌的ＰＣＲ酶联免疫吸附测定（ＥＬＩＳＡ）方法，使ＰＣＲ结果判定更加客观。方法我们使用玻璃粉吸附提纯模板ＤＮＡ，将ＰＣＲ引物５′端标记生物素，用ＰＣＲ产物与微孔板中预先包被的克隆靶基因杂交，然后用酶标链霉亲和素与杂交体中的生物素结合，最后用酶底物与所标记酶进行显色反应，通过测定其吸光度来判断结果。结果所建立的ＰＣＲＥＬＩＳＡ检测法可提高检测的灵敏度和特异性。对５０份来自结核病人高度怀疑有结核分枝杆菌的标本检测表明，ＰＣＲＥＬＩＳＡ检出２２份阳性，比抗酸染色法（７／５０）、培养法（１３／５０）和ＰＣＲ电泳法（１８／５０）检出率高，而２０份证实无结核分枝杆菌的标本，几种方法检查均为阴性。结论该法可敏感、特异和客观地检测临床标本中的结核分枝杆菌。     

聚合酶链反应检出石蜡包埋肝癌组织的乙型肝炎病毒DNA

传统免疫组织化学方法及分子杂交检测石蜡包埋肝组织乙型肝炎病毒（ＨＢＶ）ＤＮＡ灵敏度低，聚合酶链反应（ＰＣＲ）方法能否检测到石蜡肝癌组织中的ＨＢＶＤＮＡ报道尚不多见。为此，采用ＨＢＶ与丙型肝炎病毒二合一ＰＣＲ试剂盒对１７例肝癌患者的石蜡包埋癌组织进行了ＨＢＶＤＮＡ检测，结果７例阳性，阳性率为４１％，而８例非肝癌对照患者则均阴性。组织ＨＢＶＤＮＡ结果与血清ＨＢｓＡｇ比较显示，该试剂盒有较高的检出率及符合率。这为今后进一步研究大量保存的肝病石蜡包埋组织的ＨＢＶ感染提供了一个可靠灵敏的方法，也为研究其他病毒感染的石蜡包埋材料提供了参考。     

Mycobacterium tuberculosis detection by polymerase chain reaction and identification of M. tuberculosis strain

A rapid multiprimer PCR method for detection of Mycobacterium tuberculosis complex (MTC) and simultaneous identification of M. tuberculosis in clinical samples has been developed. The method is based on simultaneous amplification of two targets: a 401 bp region from the mtp40 species-specific gene sequence of M. tuberculosis and a 544 bp fragment from the RD1 genome region which is specific for MTC but absent in BCG strains. Polymerase inhibitors in this study were detected by internal control in each test. Detection sensitivity was 25 copies of M. tuberculosis genomic DNA. Seven methods for isolation of mycobacterial DNA were compared and the technique with chloroform extraction was selected as the most efficient. The proposed method was used for analysis of 37 clinical samples and the results were compared with the results of culturing, acid-fast bacilli staining, and clinical diagnosis. The method proved to be sufficiently sensitive and specific for detection of mycobacterial DNA. Moreover, in countries with only two main pathogenic species of MTC circulating (M. tuberculosis and M. bovis) this method can be used for differentiation of these two species.     

Rapid detection of Salmonella by polymerase chain reaction

Salmonella enterotoxin gene (stn) was sequenced from Salmonella enterica serotypes: Typhimurium, Typhi, Paratyphi A and B. The sequences from all the four serotypes showed complete homology with the already reported stn gene sequence of the serotype Typhimurium. As a tool for detection of this organism, four pairs of oligonucleotide primers were designed to amplify different fragments of this important pathological marker. The protocols were standardized with serotype Typhimurium in such a way so as to complete the PCR reaction in 75-90 min. These primers were found to generate specific amplicons with all the serotypes of Salmonella tested. The PCR protocols were found to be highly specific as no amplifications, specific or non-specific, were found when reactions were run using non-Salmonella DNA as template. The employment of a nested PCR markedly increased the sensitivity of the assay system in natural water samples. The protocol described herein is highly sensitive as it detects less than 10 cells of Salmonella in 250 microl of blood and approx. 1 cell in 1 ml of water without any enrichment. For the validation of this protocol, 72 coded samples of 11% skimmed milk spiked with different pathogens were received from NICED, Kolkata and analyzed for the presence of Salmonella. Our procedures detected correctly the presence of Salmonella in nine samples. 50 samples of raw milk were subjected to this PCR after enrichment for 8 h and 6 samples were found positive for Salmonella. The study indicates that Salmonella enterotoxin (stn) gene is highly conserved and the protocol devised in this study can be used as rapid and reliable method for detection of Salmonella spp. in water, milk and blood samples.     

聚合酶链反应检测痰标本中结核菌的临床应用--附284例检测结果

目的：探讨ＰＣＲ检测痰标本中结核菌诊断肺结核的价值。方法：收集１９９３年４月至１９９６年６月我科用ＰＣＲ检测２８４例肺部疾病患者痰标一中结核菌的资料与涂片及结核菌培养比较，结果：肺结核病组ＰＣＲ检测结核菌阳性率明显高于涂片（６３．８％比２４．３％）及培养法（６８．９％比３３．８％）。结论：ＰＣＲ检测痰标本中结核菌培养法灵敏性，特异性高。     

Direct detection of isoniazid-resistant Mycobacterium tuberculosis in respiratory specimens by multiplex allele-specific polymerase chain reaction

This study evaluated the feasibility of using 2 multiplex allele-specific polymerase chain reaction (MAS-PCR) assays targeting 2 mutations (codon 315 of the katG gene and the 15th nucleotide preceding the mabA-inhA operon) to directly detect isoniazid (INH)-resistant Mycobacterium tuberculosis in cultured isolates and respiratory specimens. A total of 203 M. tuberculosis isolates and 487 respiratory specimens were investigated. The MAS-PCR assays successfully amplified all M. tuberculosis isolates and acid-fast bacilli smear-positive specimens while only 49.2% of the smear-negative specimens exhibited positive MAS-PCR results. The MAS-PCR assays identified 83.4% and 79.2% of the resistant strains in the culture isolates and respiratory specimens, respectively. All the inferred genotypes were in complete accordance with subsequent DNA sequence analyses. This study suggested the application of our improved MAS-PCR protocols to provide the rapid identification of INH-resistant M. tuberculosis directly in respiratory specimens. The technical simplicity, short turnaround time, and low cost of this molecular strategy should facilitate routine diagnostic services in developing areas with a high prevalence of drug-resistant tuberculosis.     

PCR—ELISA测定结核分枝杆菌DNA临床应用评价

目的　评价聚合酶链反应 酶联免疫吸附试验 (PCR ELISA)测定结核分枝杆菌DNA临床应用价值。方法　用PCR ELISA测定 436例肺结核、172例非结核病人痰标本中的结核分枝杆菌DNA ,并结合临床诊断和痰菌检查对实验结果进行分析。结果　 436例活动性肺结核病人本法最终阳性 341例 ,阳性率 78.2 1% ,其中痰菌阳性病人本法阳性率为 97.0 7% ,痰菌阴性病人阳性率为 5 5 .33 % (10 9/ 197) ;而 172例对照患者只有 3例假阳性 ,假阳性率 1.74%。结论　本法测定痰液结核分枝杆菌DNA对肺结核的诊断具有很高的敏感性和特异性 ,尤其对痰菌阴性的活动性肺结核病人具有重要辅助诊断价值     

Real-time polymerase chain reaction detection of Neisseria meningitidis in formalin-fixed tissues from sudden deaths

Accurate identification of meningococcal sudden deaths is needed to avoid underestimation of the true incidence of the disease. This study analyzed the usefulness of a real-time polymerase chain reaction (PCR) protocol using MGB (3′-minor groove binder) probes to detect Neisseria meningitidis in formalin-fixed paraffin-embedded tissues from sudden deaths where a meningococcal fulminating infection was suspected. The protocol included detection of meningococcal DNA (ctrA gene), multiplex B/C PCR serogrouping (siaD gene), and rapid confirmation of PCR products by microcapillary electrophoresis. Sixty-nine tissues from 15 culture-confirmed meningococcal sudden deaths were analyzed (positive cases). Validation studies were performed. In each positive case, both the ctrA and the B/C siaD genes were detected. The ctrA was detected in 81.2% of the samples, whereas the serogroup (B or C) was identified in 44.9% of them. Therefore, this protocol may improve nonculture diagnosis and case ascertainment in meningococcal disease deaths, particularly when formalin-fixed tissues are the only available specimen.     

PCR检测技术在结核病诊断中的价值

用ＰＣＲ技术对１７５例临床可疑结核病，但常规培养或抗酸染色阴性的病理标本中结核菌ＤＮＡ进行了检测。结果发现有２２．３％（３９／１７５）的样本结核菌ＤＮＡ阳性。结果提示，ＰＣＲ技术在检测结核菌ＤＮＡ中具有简便、快速和灵敏的特点，其阳性结果说明被测样品中有结核菌的存在，对结核病的确诊有重要辅助意义。     

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.     

Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction

Human papilloma virus (HPV) DNA sequences have been detected in paraffin-embedded tissue using an enzymatic in vitro amplification technique known as the polymerase chain reaction. Amplification of a HPV DNA sequence before its detection with a cDNA probe significantly increases the rapidity as well as the sensitivity of detection such that a single 5-10-micron thick paraffin-embedded tissue section can be analyzed within 24 h. The assay specifically detected HPV 16 or 18 without crossreactivity with HPV 6 or 11. As few as 20 viral copies could be detected. The rapid and sensitive analysis of HPV in normal and pathological tissues using this technique may contribute significantly to identifying the role of HPV as a risk factor in carcinoma.     

Comparative evaluation of tree commercial diagnostic kits for detection of M. tuberculosis by polymerase chain reaction

Three Russian commercial PCR diagnostic kits for detection of M. tuberculosis in clinical samples are compared. The kits were evaluated by sensitivity and convenience of analysis. The specificity and sensitivity of Russian diagnostic kits are not inferior to foreign analogs, but are not devoid of some drawbacks.     

Blood and urine samples as useful sources for the direct detection of tuberculosis by polymerase chain reaction

The aim of the study was to assess the utility of the polymerase chain reaction (PCR) assay in blood and urine for the diagnosis of tuberculosis (TB). We prospectively evaluated the usefulness of PCR performed in blood and urine samples from patients with proved or probable TB compared with a control group of patients. The PCR technique was performed using IS6110 primers. We included in the study 57 patients (43 with definite TB and 14 with probable TB) and 26 controls. Blood and urine samples were drawn at the time of microbiologic diagnosis and 3, 6, 9, and 12 months later. Cultures were positive in the early period (<1 month after treatment) in 11 of 57 patients (19%) with probable or definite TB, in comparison with 42% of patients (24/57) who yielded a positive PCR (P = 0.02). Urine samples increased the sensitivity of PCR determination in blood samples by 10%. The PCR in blood and/or urine was positive in 41% of patients with pulmonary TB, in 36% of patients with extrapulmonary TB, and in 50% of patients with disseminated TB. Mycobacterium tuberculosis was still detectable by PCR in 5 of 13 patients with cured TB after 1 or more months of antituberculous treatment. The PCR detection of M. tuberculosis in blood and urine samples is useful for the diagnosis of different clinical forms of TB, mostly in those patients in which sample extraction is difficult or requires aggressive techniques. The sensitivity of this technique could be improved studying more than 1 sample in each patient, even after initiating an antituberculous treatment.     

Inosine-containing primers in human papillomavirus detection by polymerase chain reaction.

A PCR protocol has been developed using inosine-containing primers for human papilloma virus (HPV) detection. The HPV16 L1 region from CaSki-positive cells was efficiently amplified by a single reaction and directly analyzed by agarose gel electrophoresis. The method was found to be sensitive and reproducible, and it easy to use for HPV typing and screening.     

The detection of Bordetella pertussis in swab samples by polymerase chain reaction

Polymerase chain reaction (PCR) is revolutionizing the diagnosis of many infectious diseases, particularly those caused by organisms that are difficult to cultivate. Rapid and sensitive detection of Bordetella pertussis in clinical samples is important for the early diagnosis of pertussis, the prevention of transmission of the organisms and vaccine efficacy trials. In this study dacron and calcium-alginate nasopharyngeal swabs (NPSs) taken from patients with suspected pertussis were analysed by PCR using primers derived from the DNA adjacent to the B. pertussis porin gene. NPSs in Regan-Lowe medium were transported to the laboratory for bacterial culture and PCR. Chelex 100 resin was used to extract bacterial DNA from NPSs. Three hundred and five NPSs including 127 dacron and 178 calcium-alginate swabs from 165 children with suspected pertussis were tested. Fourteen (11%) dacron swabs and 10 (6%) calcium-alginate swabs were culture positive. However, 23 (18%) dacron swabs and nine (5%) calcium-alginate swabs were positive by PCR using the ethidium bromide staining method. When the digoxigenin immunoblot system was used to increase the sensitivity of PCR, 79 (62%) dacron NPSs including 14 culture-positive samples were PCR positive; however, only 28 (16%) calcium-alginate NPSs including one culture-positive sample were positive by PCR. This data indicates that PCR is more sensitive than culture and the sensitivity of PCR can be increased using the digoxigenin immunoblot system. In addition, many fewer PCR positives were found in calcium-alginate swabs compared to dacron NPSs, which may be due to the quality and the Taq polymerase inhibitory effect of calcium-alginate fibre.     

Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis.

A rapid screening test was recently established for the detection of mutations in the rpoB gene of Mycobacterium tuberculosis, a region identified as the locus for rifampin resistance (Rifr). The detection method involved the amplification by polymerase chain reaction (PCR) of the Rifr region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification products. Experience using two different PCR-SSCP formats for the evaluation of BACTEC cultures and sputum is presented here; the previously described manual procedure for the detection for the detection of radiolabelled amplification products and an automated SSCP by which fluorescein-labelled products were detected on a Pharmacia DNA sequencer apparatus. All 17 different Rifr mutations known to date were consistently detected. PCR-SSCP could be used for the evaluation of minimally grown cultures (BACTEC 12B medium with a growth index of < or = 100) and for direct screening of microscopically positive sputa with greater than 10 organisms per field (magnification, x250). Implementation of this technique could result in rapid detection of rifampin resistance in M. tuberculosis, a marker of multidrug-resistant tuberculosis.     

Development and evaluation of real-time polymerase chain reaction assays on whole blood and paraffin-embedded tissues for rapid diagnosis of human brucellosis

This study aimed at developing a real-time polymerase chain reaction (PCR) assay for the rapid diagnosis of human brucellosis on clinical specimens. Three assays with hybridization probe detection on the LightCycler instrument were developed and compared targeting the 16S-23S internal transcribed spacer region (ITS) and the genes encoding for omp25 and omp31. All assays showed 100% analytical sensitivity and 100% specificity when tested on 28 consecutive clinical isolates of Brucella sp. and 19 clinical isolates of common Gram-negative and Gram-positive bacterial pathogens, respectively. The ITS assay was the most sensitive with a limit of detection of 2 genome equivalents per PCR reaction. This assay was then clinically validated prospectively with 354 samples (351 whole blood samples and 3 paraffin-embedded tissues) collected from 340 patients, 24 samples from patients with active brucellosis including 2 relapsing cases, 31 with treated brucellosis, and 299 seronegative patients where brucellosis was initially suspected. The clinical sensitivity, specificity, and positive and negative predictive values of the ITS assay were 66.7%, 99.7%, 94.1%, and 97.6%, compared with culture at 77%, 100%, 100%, and 97.3%, respectively. The difference in sensitivity between culture and ITS-PCR was not statistically significant (P = 0.71). Both relapsing cases were PCR positive. Treated patients were PCR negative. All 3 assays were positive on tissue samples, but the omp25 and omp31 assays did not detect Brucella sp. DNA in blood samples. Because omp31 is not present in Brucella abortus, these data indicate that the 28 tested isolates are most likely Brucella melitensis. ITS-PCR is rapid and could augment the clinical laboratory diagnosis of human brucellosis.     

多重聚合酶链反应检测生殖支原体感染

目的建立一种敏感快速的多重聚合酶链反应（ＰＣＲ）用于检测生殖支原体（Ｍｇ）。方法性病门诊患者５１１例，正常对照５６人。分泌物（女性为宫颈、男性为尿道拭子）标本用ＮＰ－４０裂解液一步法制备模板ＤＮＡ。设计Ｍｇ种属特异性引物及功能性结蛋白基因引物，先进行标准株的特异性与敏感性试验，然后进行临床标本的ＰＣＲ扩增反应。结果两对引物有良好的特异性，对Ｍｇ标准株模板敏感性可达１０－６μｇ。临床标本检测，广东地区２６１例性病门诊患者的Ｍｇ－ＤＮＡ阳性率（５４／２６１，２０．７％）高于上海（９／８４，１０．７％）、常州（８／７０，１１．４％）及南京地区（１１／９６，１１．５％；χ２＝１６．１，Ｐ＜０．０１），后三者的感染率互相间比较，差异无显著性（χ２＝０．０５６，Ｐ＞０．０５）。性病门诊患者５１１例的Ｍｇ－ＤＮＡ总阳性率（８２／５１１，１６．１％）与５６例正常对照者（１／５６，１．８％）比较，差异有非常显著性（χ２＝７．８８２，Ｐ＜０．０１）。结论建立的ＰＣＲ技术检测Ｍｇ具有敏感、快速、特异的特点，是一种可靠的实验诊断手段。     

磁捕法对传染性非典型肺炎冠状病毒RNA富集体系的初步研究

目的 初步研究建立磁捕法对传染性非典型肺炎又称严重急性呼吸综合征(SARS)冠状病毒RNA的富集、纯化体系,以提高逆转录聚合酶链反应(RT-PCR)检测SARS冠状病毒RNA的敏感性。方法 根据国际基因库公布的SARS冠状病毒基因序列,自行设计引物、探针,体外克隆目的RNA片段作为标准物质;利用标准RNA以及模拟SARS血清标本的RNA,评价自行设计的SARS冠状病毒巢式RT-PCR检测体系和磁捕法SARS冠状病毒RNA富集检测体系的检测效果。结果SARS冠状病毒巢式RT-PCR检测体系,对于模拟血清SARS标本RNA的最低检测样本浓度为20拷贝/μl,而磁捕法SARS冠状病毒RNA富集检测体系的最低检测样本浓度为1拷贝/μl。结论 初步建立的磁捕法SARS冠状病毒RNA富集检测体系,可有效地对低浓度的RNA样本进行浓缩、纯化,有效提高SARS RT-PCR检测方法的敏感性。