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1.
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.  相似文献   

2.
We evaluated the use of culture and PCR-based assay for the direct detection of Mycobacterium tuberculosis (MTB) from sputum collected and stored on filter paper at room temperature for 5 days; the results were compared with those of staining and conventional culture of fresh sputum before storage (the 'gold standard'). Out of 231 sputum specimens examined, MTB was recovered from 124 samples by culture before storage. The culture positivity rate was significantly decreased to 70% after 5 days storage. For PCR assay, a fragment of 377 bp of the IS6110 sequence was amplified and detected using three methods: first PCR product combined with agarose gel electrophoresis (AGE); first PCR product with dot blot hybridization (DBH); nested PCR with AGE. Compared with culture, the sensitivity, specificity, and efficiency for first PCR with AGE were 71.8, 100 and 84.9% respectively; PCR with DBH gave results of 89.5, 96.3 and 92.6% respectively; the same values for nested PCR were 96.0, 97.2, and 96.5% respectively. Of these three methods, nested PCR gave excellent sensitivity and specificity with no significant difference (p = 0.727) from conventional culture. The storage of sputum on filter paper and storage at room temperature for 5 days had no apparent effect on the performance of nested PCR. We propose that this collection and storage method be considered for transporting sputum specimens from peripheral health centers or from the field; specimens may be sent by post to a central point for both culture and PCR analysis by trained technicians supervised in accordance with a well-established quality control system.  相似文献   

3.
Prior studies have shown that Onchocerca volvulus DNA can be detected in skin snips and in black flies after polymerase chain reaction (PCR) with primers specific for repeated "O-150" DNA sequences. We have adapted a paper chromatography hybridization assay (PCHA) to detect amplified O-150 DNA and compared this method to two established methods, namely agarose gel electrophoresis (AGE) and hybridization enzyme-linked immunosorbent assay (ELISA). The minimum amounts of purified O-150 DNA detected by PCHA, AGE, and ELISA were 5, 10, and 2 ng, respectively. The three methods had similar estimated sensitivities for detecting O. volvulus DNA amplified from skin snips from African subjects with onchocerciasis (88%, 84%, and 91%, respectively). No false positive results were observed with skin snips from uninfected control subjects. The paper chromatography hybridization assay detects PCR products in 30 minutes without electricity or special equipment. This technology brings DNA detection a step closer to widespread use in field settings.  相似文献   

4.
微孔杂交技术诊断肺外结核   总被引:1,自引:0,他引:1  
目的 将聚合酶链反应(PCR)、核酸杂交、酶联免疫吸附三种技术有机结合,从分子水平检测结核杆菌,为临床诊断肺外结核提供病原学依据。方法 使用生物素标记的特异性引物扩增结核杆菌(TB)DNA,带有生物素的产物与包被在微孔板内的靶基因杂交,加入酶标链毒亲和素与生物素结合,最后加辣根过氧化物酶显色,2MH2SO4终止反应。根据酶标仪检测各孔吸光度判断结果。结果通过对临床高度怀疑为肺外结核的408份标本的检测,PCR微孔板杂交法的检出率为43.6%,培养法的检出率为25.9%直接涂片抗酸染色为6.4%。结论 PCR杂交法灵敏度高,特异性强,有助于临床准确、快速诊断肺外结核。  相似文献   

5.
目的 探讨快速检测rpoB基因突变的敏感方法 ,以期建立适合我国国情的结核分支杆菌耐药株的快速检测手段。方法 根据结核分支杆菌野生株序列 ,自行设计覆盖rpoB基因核心突变区的系列寡核苷酸探针并将其固定在尼龙膜上 ,然后应用生物素标记的特异性引物扩增包含核心突变区的rpoB基因片段 ,与固定在膜上的寡核苷酸探针杂交 ,对突变位点进行快速检测 ,并与药敏试验及DNA测序结果进行比较。结果 应用反向斑点杂交法检测 36株耐药株和 2 2株敏感株rpoB基因的突变位点 ,并据此判断其对利福平的药敏特性 ,结果敏感性为 88.9% ,特异性为 86 .4 % ,药敏结果符合率为 87.9% ,与测序结果的符合率为 89.7%。结论 反向斑点杂交法可以快速、敏感地检测rpoB基因突变 ,进而早期判断结核分支杆菌对利福平的药敏特性。  相似文献   

6.
An in-house sensitive and easy-to-use solid-phase enzyme-linked immunoassay (ELISA) was adapted for the detection and quantification of hepatitis B virus (HBV) PreS1 envelope antigen in serum, and compared with the HBV DNA Hybrid Capture system from Murex and the polymerase chain reaction (PCR) Amplicor HBV Monitor assay from Roche. Twenty-five patients with chronic hepatitis B after liver transplantation were included in this study. The sensitivity of our ELISA was found to be 50 pg of HBsAg/PreS1Ag ml–1. The linearity was between 0.1 and 100 ng ml–1. Intra-assay reproducibility was obtained with a standard deviation of <1%. No correlation between the presence of serum PreS1 antigen and viral DNA detected by direct hybridization (Murex) was observed. In contrast, there was a significant 96% correspondence in the presence of PreS1 antigen and viral DNA detected and quantified by the PCR assay (Roche). In conclusion, the most important and reliable markers for monitoring residual HBV replication in serum were HBV DNA by the PCR assay, and virus envelope PreS1Ag by our in-house ELISA. Thus, PreS1Ag disappearance in serum could be used for evaluating the efficacy of antiviral therapies.  相似文献   

7.
目的评估涂片、培养、PCR和增菌PCR检测结核分枝杆菌临床应用价值。方法对124例临床确诊的肺结核、可疑结核患者和非结核病人痰标本的涂片、培养、PCR和增菌PCR四种方法的检测结果进行比较。结果涂片、培养、PCR和4及7d的增菌PCR检测31例临床确诊的肺结核病人痰标本阳性率分别为22.5%、32.2%、54.8%、64.5%和87.1%;检测59例临床可疑肺结核病人痰标本阳性率分别为13.6%、18.6%、28.8%、37.3%和52.5%。比较四种方法的阳性检测率有显著性差异(P<0.05)。检测34例非结核病人痰标本,涂片、培养均为阴性,而PCR和增菌PCR均有1例假阳性,假阳性率2.9%。比较PCR与增菌PCR对菌阳和菌阴病人的痰标本阳性检测率,有显著性差异(P<0.05),而两种方法的假阳性率相同。结论增菌PCR检测结核分枝杆菌具有很高的敏感性和特异性,可作为结核病的有效辅助诊断方法之一。  相似文献   

8.
结核性腹膜炎的实验室诊断   总被引:27,自引:0,他引:27  
目的评价聚合酶链反应(PCR)结合Southern杂交技术及酶联免疫吸附试验(ELISA)对结核性腹膜炎的诊断价值。方法用PCR结合地高辛标记核酸探针Southern杂交技术检测42例结核性腹水中结核分支杆菌DNA,并与常规细菌学检测及ELISA对比。引物来自结核分支杆菌特异重复插入序列IS6110。特异性通过杂交及限制性内切酶SalⅠ酶切证实。同时比较了Southern杂交检测与凝胶电泳检测的敏感性。结果PCR的敏感性为69%,ELISA为71%,培养为9%,涂片镜检均为阴性。并发现杂交较凝胶电泳检测更敏感。结论PCR和ELISA法对结核性腹膜炎有较高的诊断价值,但前者更具有特异性。将Southern杂交技术与PCR技术结合,可进一步提高检测的敏感性和特异性。  相似文献   

9.
目的探讨巢式实时荧光定量PCR(QNRT-PCR)检测结核分枝杆菌(MTB)DNA的临床价值。方法应用SYBR Green I建立检测结核分枝杆菌MTP64基因的QNRT—PCR方法,分别检测24份肺结核患者痰液,27份结核性胸膜炎患者胸水,以及对照组75份痰液和胸水的MTBDNA含量。结果以培养为金标准,QNRT—PCR敏感性、特异性、阳性预测值(PPV)、阴性预测值(NPV)分别为95.83%、61.54%、69.70%和94.12%,结核性胸膜炎的阳性率为70.37%。三种PCR方法阳性率比较,差异具有统计学意义(P〈0.05),QNRT—PCR与实时荧光定量PCR拷贝数配对检验差异没有统计学意义(P〉0.05),QNRT—PCR与实时荧光定量PCR的Cr值配对检验差异具有统计学意义(P〈0.01)。结论QNRT—PCR方法可检测痰液和胸水MTB DNA,对含微量MTB DNA样本的高敏感性在结核病的早期诊断中有重要参考价值。  相似文献   

10.
目的探讨应用十二烷基硫酸钠(SDS)处理标本在结核分支杆菌聚合酶链反应(PCR)中的价值。方法从186例活动性结核病人收集痰、胸腹水、脑脊液、尿、血液等标本266份,标本分别用SDS和常规方法进行处理。两种方法处理的标本同时用PCR-反相膜杂交试验检测结核分支杆菌,并将结果进行比较。结果用SDS和常规方法处理的标本,PCR-反相膜杂交试验结核分支杆菌的检出率分别为65.0%(173/266)和51.5%(136/266)。通过配对计数资料卡方检验,SDS法和常规法之间的差异具有非常显著性意义(χ  相似文献   

11.
Rapid diagnosis of pulmonary tuberculosis by mycobacteriophage assay.   总被引:1,自引:0,他引:1  
We evaluated FASTPlaqueTB, a recently introduced bacteriophage assay for rapid detection of Mycobacterium tuberculosis complex in sputum specimens, using 169 non-duplicate sputum specimens from patients suspected of pulmonary tuberculosis. The results of 160 specimens were analysed. FASTPlaqueTB assay detected tuberculosis in 77% (46/60) of culture-positive cases. Among the AFB smear-positive cases (n = 47) it had a sensitivity of 76% and specificity of 60% while among AFB smear-negative cases (n = 113) its sensitivity and specificity were 78% and 98%, respectively. The overall sensitivity and specificity of the technique were 77% and 96%, respectively, and the positive and negative predictive values were respectively 92% and 87%. The overall efficiency of the test was 89%. Test results were available in 48 h.  相似文献   

12.
rDNA探针杂交检测病人痰标本中结核杆菌的研究   总被引:2,自引:0,他引:2  
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份结核病人,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bPCR扩增产物的敏感性为100pg。rDNA探针与受试24种分枝杆菌和11种非分枝杆菌引物bPCR扩增产物杂交只有结核分枝杆菌、胃分枝杆菌为阳性杂交,特异性较高。而rDNA探针对90份结核病人痰标本引物b扩增产物检测的阳性率为80.2%,高于PCR扩增产物电泳检测结果 (64%)。rDNA探针与30份非结核病人痰标本杂交结果均为阴性杂交。结论 rDNA探针与引物bPCR扩增相结合能提高结核病人痰标本检测的敏感性与特异性。  相似文献   

13.
目的 评价rRNA扩增方法 在临床应用的效果。 方法 选取到北京胸科医院、山东省胸科医院、河南疾控中心结核病防治所3个机构就诊的肺结核可疑症状者及健康志愿者的痰标本为研究对象,共纳入551例痰标本,对每例痰标本均进行涂片显微镜检查、罗氏培养、rRNA扩增试验以及实时荧光定量PCR。与罗氏培养方法 比较分析rRNA扩增方法 的敏感性、特异性、阳性预测值和阴性预测值以及与实时荧光定量PCR扩增方法 的一致性。 结果rRNA扩增方法 的敏感性为98.5%,特异性为95.0%,阳性预测值为95.0%,阴性预测值为98.5%,rRNA扩增方法 在涂阳标本和涂阴标本间的敏感度和特异度差异均有统计学意义(χ2=9.60,P=0.002;χ2=79.80,P<0.01)。与PCR检测结果的一致性为93.8%,涂阳和涂阴标本中2种方法 的一致性差异有统计学意义(χ2=4.45,P=0.035)。 结论 rRNA扩增方法 的敏感度和特异度均较好,且能明显缩短诊断时间,该方法 是一种较有前景的结核病实验室诊断方法 。  相似文献   

14.
SETTING: The polymerase chain reaction (PCR) is a highly sensitive method for the detection of Mycobacterium tuberculosis and is available in most countries, though to a lesser extent in rural areas. OBJECTIVE: To amplify M. tuberculosis DNA sequences of sputum spotted on FTA cards and compare them with the results of microscopic examination among culture-positive samples. DESIGN: A total of 102 sputum specimens of TB patients in treatment were spotted on FTA cards and stored at room temperature until DNA analysis. We assessed the IS6110 region of M. tuberculosis. The efficacy of the PCR assay for the direct detection of M. tuberculosis was evaluated and compared with the results of cultures (Middlebrook 7H9 broth) and smears of fresh sputum specimens. RESULTS: We were able to detect 10 fg/microl of mycobacterial DNA even after 6 months in storage. The PCR sensitivity and specificity using the FTA card system were 82% and 96%, while microscopic examination showed 41% and 95%, respectively. CONCLUSION: The FTA card system for the storage of bacterial DNA from sputum samples should be considered for the molecular diagnosis of tuberculosis. Samples can easily be obtained from geographically isolated populations and shipped by mail for accurate molecular diagnosis.  相似文献   

15.
目的建立一种特异、灵敏、重复性好的鹦鹉热嗜性衣原体的TaqMan-MGB荧光定量PCR检测方法。方法利用鹦鹉热嗜性衣原体MOMP基因的特异保守序列设计引物和MGB探针,通过优化,获得最佳反应体系与反应条件,同时进行特异性、重复性、灵敏性评价与Spike-test实验进行临床应用性评价,利用该检测方法对禽鸟类粪便样品进行检测,并与常规PCR检测方法进行比较。结果该方法在0.01pg~100pg范围内线性相关系数为0.999,扩增效率为97.7%;对鹦鹉热嗜性衣原体菌株检测结果均呈现阳性,而非鹦鹉热嗜性衣原体菌株及其它衣原体菌株为阴性;重复性试验中,变异系数为0.317%~0.563%;检测灵敏性为0.01pg;Spike-test试验中最低检出量为25个EB;该方法对禽鸟类粪便样品的检出率为14.3%(40/282),高于常规PCR检测法的7.4%(21/282)。结论本文所建立的Taqman-MGB荧光定量PCR检测法是一种灵敏、高效、稳定,可在嗜性衣原体属中准确检测鹦鹉热嗜性衣原体的方法,该方法的建立更有意于今后对禽鸟类实施鹦鹉热的临床诊断与分子流行病学研究。  相似文献   

16.
The hybridization assay using polymerase chain reaction (PCR) is useful for rapid detection of Mycobacterium tuberculosis (M. tuberculosis). A 77-year-old female was admitted to our hospital complaining of cough, and examination of the sputum culture showed M. tuberculosis. Her chest X-ray showed a variety of abnormal shadows, such as a cavity lesion, multiple coin lesions, and infiltrates. Malignant disease was also suspected to be involved, with the complication of pulmonary tuberculosis. Specimens were obtained by transbronchial lung biopsy (TBLB) from coin lesions. The hybridization assay using PCR on the TBLB specimens showed M. tuberculosis gene expression. She was treated with anti-tuberculous drugs. All shadows in her chest X-ray were improved six months after admission. She was remained well without recurrence for more than two years after admission. The hybridization assay using PCR with TBLB specimens is useful for the detection of M. tuberculosis.  相似文献   

17.
目的 探讨PCR -荧光双标记探针杂交定量结核杆菌技术对肺结核的诊断价值并进行卫生经济学评价。方法 对 2 0 0 2年 5月至 2 0 0 2年 11月收治的 30 1例患者进行回顾性研究 ,将痰PCR-荧光探针杂交法与荧光抗酸染色涂片法进行比较 ,以综合临床资料最后确诊为诊断标准 ,计算两种方法的诊断效率及进行成本 -效果分析。结果 痰PCR -荧光探针法诊断肺结核的灵敏度为5 1.9% ,涂片法 1次、3次、6次的灵敏度分别为 4 9.2 %、5 7.4 %、6 1.2 % ,经统计学分析 ,两种方法的检出率比较差异无显著性 (P>0 .0 5 ) ,进行成本 -效果分析 ,每检出 1例肺结核行 1次痰PCR -荧光探针法患者要花费 2 5 3.4 7元 ,行 1次涂片法要花费 5 0 .17元。结论 PCR -荧光探针杂交法与涂片法比较同样具有快速、有效等优点 ,但经济效率低于荧光涂片法 ,目前尚不能取代后者在诊断结核病中的传统地位。  相似文献   

18.
目的:比较噬菌体裂解法与BACTEC-460法在结核分支杆菌快速检测及鉴定中的价值。方法:应用噬菌体裂解法和BACTEC-460法分别检测30株结构分支杆菌临床分离株、10株非结核分支杆菌和7株非分支杆菌,以及60份临床确诊的肺结核患者的痰标本,结果:所有结核分支杆菌临床分离株噬菌体裂解试验均为阳性,而非结核分支杆菌和非分支杆菌均为阴性。41份BACTEC-460培养阳性和19份BACTEC-460培养阴性的痰标本中,噬菌体裂试验分别有34份(82.9%)和5份(26.3%)阳性。结论:噬菌体裂解法可以简便,快速地检测结核分支杆菌,并且具有较高的敏感性的特异性。  相似文献   

19.
目的评价聚合酶链反应(PCR)方法对结核性腹膜炎的诊断价值。方法用PCR技术检测30例结核腹水中结核分支杆菌DNA,并与腹水涂片抗酸染色及酶联免疫吸附试验(ELISA)检测腹水中抗PPD抗体进行比较。结果PCR的阳性率为60%,特异性94.4%;ELISA法阳性率63.3%,特异性72.2%;涂片镜检均为阴性。结论PCR在诊断结核性腹膜炎具有较高的敏感性和特异性,优于ELISA法及涂片镜检,如与ELISA技术结合可进一步提高检测的敏感性和特异性。  相似文献   

20.
目的 比较各种DNA 固定方法对结核分枝杆菌PCR 产物杂交结果的影响?方法 选取结核分枝杆菌PCR 扩增产物并点于尼龙膜上,用紫外线?加热和碱液三种方法固定DNA,然后以PCR 产物作探针用增强化学发光标记试剂进行标记,并对三种不同方法固定的PCR 产物杂交分析?结果 结核分枝杆菌DNA固定方法对PCR 扩增产物的检测结果具有较大的影响,碱液固定具有较好的敏感性和重复性?结论 各地结核分枝杆菌PCR 实验室可考虑用碱液法对PCR 产物固定进而进行杂交分析。  相似文献   

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