聚合酶链反应在结核性胸膜炎和结核性腹膜炎中的应用

应用聚合酶链反应技术对６６例胸水和腹水进行结核ＤＮＡ检测。１７例结核性胸腹水ＰＣＲ检测阳性率为５２．９４％，４９例非结核性胸腹水未出现假阳性结果。结果表明ＰＣＲ直接检测胸腹水中结核菌显示出快速、敏感和高效等优点。同时对影响ＰＣＲ检测结核菌的某些因素作了分析。     

多聚酶链式反应速检测肺活检组织中的结核杆菌DNA

利用多聚酶链式反应（ＰＣＲ）以３１例病理可疑诊断为结核的肺活检切片进行结核菌ＤＮＡ的检测，并与抗酸染色进行了比较。用蛋白酶Ｋ和冻融融解法快提切片ＤＮＡ。３１例中有２４例ＰＣＲ阳性，１４例抗酸染色阳性；其中１７例抗酸色性经ＰＣＲ检出１２例阳性，１４例抗酸染色阳性ＰＣＲ检出１２例阳性。ＰＣＲ检测阳性率明显高于抗酸染色（Ｐ〈０．０１）。说明ＰＣＲ用于活检组织诊断理一快速、敏感性较高、特异性较强的技术。此     

多聚酶链式反应快速检测肺活检组织中的结核杆菌DNA

利用多聚酶链式反应（ＰＣＲ）对３１例病理可疑诊断为结核的肺活检切片进行结核菌ＮＤＡ的检测，并与抗酸染色进行了比较。用蛋白酶Ｋ和冻融裂解法快提切片中ＤＮＡ。３１例中有２４例ＰＣＲ阳性，１４例抗酸染色阳性；其中１７例抗酸染色阴性经ＰＣＲ检出１２例阳性，１４例抗酸染色阳性ＰＣＲ检出１２例阳性。ＰＣＲ检测阳性率明显高于抗酸染色（Ｐ＜０．０１）。说明ＰＣＲ用于肺活检组织诊断结核是一快速、敏感性较高、特异性较强的技术。此外，对影响ＰＣＲ结果的因素进行了分析。     

PCR在结核病研究中应用

本文综述了ＰＣＲ技术在结核病临床研究中的新进展，对如何选择引物、处理标本、减少假阳性和假阴性等进行了详细说明，对ＰＣＲ技术在结核菌菌种鉴定、流行病学研究和结核耐药基因检测等方面应用进行了阐述。     

PCR技术在儿科结核病诊断中存在的问题

观察多聚酶链反应技术在结核病临床诊断上的应用以及存在的问题，方法：２２例结核病符合临床诊断标准，５０例对照组，应用ＰＣＲ技术，经凝胶电泳法检测结果。结果血样本ＴＢ－ＤＮＡ－ＰＣＲ的阳性率为结核组１８．１８％，对照组３６．００％，敏感度１８．１８％，特异性６４．００％，假阳性３６．００％，假阴性８１．８２％。     

细菌DNA的聚合酶链反应扩增及反相杂交初步分型

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的细菌ＤＮＡ。结果对２４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，与人类基因组ＤＮＡ、真菌及病毒无交叉反应；２２例血培养阳性标本及４例脑脊液培养阳性标本均扩增出３７１ｂｐ长度ＤＮＡ条带，反相杂交法区分革兰阳性／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，具有特异、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     

聚合酶链反应凝胶呈像技术检测乙型肝炎病毒

目的建立聚合酶链反应（ＰＣＲ）凝胶呈像（ｇｅｌｄｏｃｕｍｅｎｔａｔｉｏｎｓｙｓｔｅｍｓ，ＧＤＳ）技术，并应用于临床检测乙型肝炎病毒（ＨＢＶ）核酸ＤＮＡ。方法用凝胶呈像技术对血清ＨＢＶＤＮＡＰＣＲ扩增产物的电泳凝胶进行摄像、分析，并与紫外灯下的肉眼观察结果相比较，对肉眼未判断出阳性而凝胶呈像分析出阳性的血清标本，用定量ＰＣＲ（ＱＰＣＲ）方法检测ＨＢＶＤＮＡ。结果通过３８份血清标本的检测，发现用凝胶呈像技术判断的结果比单纯肉眼观察的阳性率高。前者１９份阳性（５０％），后者１６份阳性（４２．１％），符合率为９２．１％。经χ２检验（χ２＝０．４７７，Ｐ＞０．０５），说明两种方法有很好的一致性。凝胶呈像分析为阳性，而肉眼观察阴性的３份血清标本，经定量ＰＣＲ检测为阳性。结论应用凝胶呈像技术比ＰＣＲ产物电泳后紫外灯下肉眼观察的灵敏度高、客观性强，能避免与紫外线接触，并可长期保留检测结果。     

PCR扩增血液中结核杆菌DNA的研究

ＰＣＲ扩增血液中结核杆菌ＤＮＡ的研究东台市人民医院（２２４２００）尹洪波，刘国栋作者应用ＰＣＲ技术检测３８例血液中的结核杆菌，并进行临床分析。材料和方法一、研究对象均排除ＨＩＶ和肺外结核病３８例，其中１６例活动型肺结核分为Ａ组１２例，为痰涂片阳性未治...     

聚合酶链反应检测结核菌的应用研究

采用３８ＫＤ－４１９ｂｐ系统检测１０９８例病人的标本，并同时进行涂片找结核菌、结核菌培养等检查。结果表明，结核病ＰＣＲ检测的阳性率为５４．７％（４２８／７８２），菌阳率为２７．１％（１９０／７８２），说明ＰＣＲ是敏感性和特异性均较好的方法。在实验过程中使用了简易的热起动法，同时限定了敏感度，设立一系列的质控，并对影响ＰＣＲ检测结核菌的各种因素作了分析。     

聚合酶链反应凝胶呈像技术检测乙型肝炎病毒

目的 建立聚合酶链反应（ＰＣＲ）－凝胶呈像（ｇｅｌｄｏｃｕｍｅｎｔａｔｉｏｎｓｙｓｔｅｍＧＤＳ）技术,并应用于临床检测乙型肝炎病毒（ＨＢＶ）核酸ＤＮＡ。方法 用凝胶呈像技术对血清ＨＢＶ－ＤＮＡＰＣＲ扩增产物的电泳凝胶进行摄像,分析,并与紫外灯下的肉眼观察结果相比较,对肉眼未判断出阳性而凝胶呈像分析邮阳性的血清标本,用定量ＰＣＲ（ＱＰＣＲ）方法检测ＨＢＶ－ＤＮＡ。结果 通过３８份血清标本的检测,发现     

聚合酶链反应扩增结核杆菌的临床应用

采用PCR技术扩增人型结核分枝杆菌特异的158 bp DNA片段靶基因,检测60例肺结核病人痰标本,结果表明,阳性率达58.3%,对照的常规培养法阳性率为11.7%。20例非结核病患者痰标本两种检测结果均为阴性.研究结果表明,PCR 扩增检测结核杆菌技术比常规检验方法快速敏感,且有较高的特异性。     

Detection of mycobacterial DNA in papulonecrotic tuberculid lesions by polymerase chain reaction

Tuberculids are a heterogeneous group of cutaneous lesions. Recent discoveries of M. Tuberculosis DNA in these lesions by PCR suggest that M. tuberculosis could play a role in their pathogenesis. The aim of this study was to demonstrate the presence of M. tuberculosis DNA by polymerase chain reaction in papulonecrotic tuberculid lesions. Skin biopsy specimens from ten patients with papulonecrotic tuberculid lesions (histopathologic features) were studied. All of them tested solidly positive in a tuberculin intradermal test. A gene-amplification PCR, using primers capable of amplifying DNA in the M. tuberculosis complex, was performed to detect M. tuberculosis DNA in the lesions. A 285-bp sequence specific of M. tuberculosis complex was amplified and confirmed by Southern-blot hybridation with a 32 p 5'-labelled internal probe. No inhibitors were detected in the negative PCR samples. The PCR technique makes the detection of mycobacterial DNA in tuberculids a possibility, and therefore provides a rational basis for antituberculous therapy and for the clinical management of these disorders.     

实时荧光定量PCR检测血浆结核分枝杆菌DNA的初步临床应用

目的 评价实时荧光定量PCR技术检测血浆（或血清）结核分枝杆菌（Mycobacterium tuberculosis,MTB）DNA的技术。方法 建立实时荧光定期量PCR方法测定血浆MTB DNA,分别检测临床确诊的55例结核病患者血清43份,血浆25份以及非结核肺部疾病患者血清18份,健康体检血清18份和健康体检者血浆11份的MTB DNA含量。结果 18例非结板肺疾病患者血清、18例健康体检者血清以及11例健康体检者血浆MTB DNA全部阴性。55例初诊结核患者中10例（18．2％）治疗前血浆（或血清）MTB DNA阳性。在痰涂片阴性的18例结核患者中,血浆（或血清）MTB DNA阳性检出率为27．8％（5／18）,其特异性达100％。结论 本研究证实了结核患者血浆（或血清）循环MTB DNA的存在,其定量检测对痰涂片阴性及无痰患者的结核病诊断有重要参考价值。     

Rapid direct detection of multiple rifampin and isoniazid resistance mutations in Mycobacterium tuberculosis in respiratory samples by real-time PCR

Rapid detection of resistance in Mycobacterium tuberculosis can optimize the efficacy of antituberculous therapy and control the transmission of resistant M. tuberculosis strains. Real-time PCR has minimized the time required to obtain the susceptibility pattern of M. tuberculosis strains, but little effort has been made to adapt this rapid technique to the direct detection of resistance from clinical samples. In this study, we adapted and evaluated a real-time PCR design for direct detection of resistance mutations in clinical respiratory samples. The real-time PCR was evaluated with (i) 11 clinical respiratory samples harboring bacilli resistant to isoniazid (INH) and/or rifampin (RIF), (ii) 10 culture-negative sputa spiked with a set of strains encoding 14 different resistance mutations in 10 independent codons, and (iii) 16 sputa harboring susceptible strains. The results obtained with this real-time PCR design completely agreed with DNA sequencing data. In all sputa harboring resistant M. tuberculosis strains, the mutation encoding resistance was successfully detected. No mutation was detected in any of the susceptible sputa. The test was applied only to smear-positive specimens and succeeded in detecting a bacterial load equivalent to 10(3) CFU/ml in sputum samples (10 acid-fast bacilli/line). The analytical specificity of this method was proved with a set of 14 different non-M. tuberculosis bacteria. This real-time PCR design is an adequate method for the specific and rapid detection of RIF and INH resistance in smear-positive clinical respiratory samples.     

Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears

We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB.     

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.     

Detection of Mycobacterium tuberculosis DNA in respiratory and nonrespiratory specimens by the Amplicor MTB PCR

To evaluate the diagnostic performance of a commercially available Mycobacterium tuberculosis PCR assay (Amplicor MTB-ROCHE), 2296 respiratory and nonrespiratory specimens from 2296 patients with suspected tuberculosis (TB) were collected prospectively in an 8-year period. Clinical data for each patient were abstracted, and all samples were examined blindly by direct microscopy, culture, and PCR. M. tuberculosis DNA was detected in 93 of 113 culture-positive samples and in 29 of 38 samples from patients with probable TB. The lowest sensitivity was observed in pleural fluid and abscess aspirates. The sensitivity, specificity, and positive predictive value of the assay were 97.2%, 100%, and 100% for smear-positive specimens and 75.3%, 97.0%, and 47.5% for smear-negative specimens, respectively. The PCR cost per additional correct clinical decision was Euro 2826 but would have declined to Euro 308 if the test was applied only to smear-positive specimens. The overall performance of Amplicor MTB test was excellent for smear-positive, but suboptimal for smear-negative specimens.     

DNA芯片快速检测结核分枝杆菌利福平耐药rpoB基因突变

目的建立快速检测结核分枝杆菌利福平（RFP）耐药rpoB基因突变的DNA芯片。方法从34株临床痰样本中提取结核分枝杆菌的基因组DNA，采用聚合酶链反应（PCR）扩增含有rpoB基因突变位点的特异DNA片段，荧光标记后与芯片上含有特异突变位点的寡核苷酸探针进行杂交，同时与DNA测序法比较。结果用DNA芯片检测出19株耐药株和15株敏感株，且结果与DNA测序相同，准确率可达88．2％，敏感性为78．9％，特异性为100．0％。结论DNA芯片快速检测结核分枝杆菌对RFP的耐药rpoB基因突变是一种操作简便、准确性、敏感性和特异性较高，且易于开展的方法。     

Methods of extraction of Mycobacterium tuberculosis DNA from clinical samples for PCR: comparison and evaluation

Several commonly used methods of extraction of Mycobacterium tuberculosis DNA from clinical samples for further use in PCR were compared. We used the commercial sets "DNA-express" (Litekh, Russia) and "DNA-sorb-B" (Amplisens, Russia) as well as phenol extraction, fast alkaline lysis and DNA sorption on silicone gel for DNA extraction. All methods were compared by the quantity of isolated DNA and stability of DNA preparation as well as by efficiency and cost of extraction. "DNA-express" proved to be most effective in the extraction of Mycobacterium tuberculosis DNA with its cost being the smallest.     

通过增加痰液量和超声裂菌提高结核分枝杆菌荧光定量PCR检出率

目的通过增加痰液量和超声破碎法提取痰液中的结核菌DNA,提高结核分枝杆菌实时荧光定量聚合酶链反应(PCR)的检出率。方法选择206例肺结核患者,以103例非结核患者作为对照组;肺结核患者同时进行结核菌涂片,并在美国BD公司的MAGIT960仪器上进行液体快速培养结核菌。另收集至少5 mL的痰液,加入2~3倍体积的4%氢氧化钠液化后,取1 mL按照采用常规方法抽提痰液中的结核菌DNA,剩余部分采用改进的超声破碎法提取结核菌DNA;两者同时进行实时荧光定量PCR检测。结果采用增量超声法,对于涂片阳性、培养阳性的结核患者,阳性检出率由87.5%(可信区间为81.4%~93.6%)提升至95.5%(可信区间为91.7%~99.4%)(P0.05)。对于涂片阴性、培养阳性的患者,阳性检出率由57.4%(可信区间为47.5%~67.4%)提高至83%(可信区间为75.4%~90.6%)(P0.01)。增量超声法比常规方法,定量值平均提高14倍以上。结论通过增加痰液量和超声破碎法提取痰液中的结核菌DNA,可提高实时荧光定量PCR结核分枝杆菌的检出率,值得进一步推广应用。