聚合酶链反应快速诊断结核病的应用研究

应用聚合酶链反应（ＰＣＲ）技术建立了扩增结核杆菌特异重复序列ＩＳ６１１０部分基因的方法。对９种抗酸分枝杆菌、３种普通菌进行检测，结果仅人型结核杆菌、牛型结核杆菌及ＢＣＧ扩增出１２３ｂｐ特异性条带，ＰＣＲ产物经ＳａｌⅠ酶切后产生７０ｂｐ与５３ｂｐ两个片段。ＰＣＲ检测人型结核杆菌的敏感性达１０ｆｇＤＮＡ或５个菌体。应用ＰＣＲ检测了１０９份结核临床标本，其总阳性率为７２．５％，明显高于抗酸染色涂片（２．８％）与细菌培养（９．２％）的阳性率（Ｐ＜０．００１）。ＰＣＲ的检出率也较ＥＬＩＳＡ法检测抗ＰＰＤ－ＩｇＧ的阳性率（６３．３％）为高，但尚无统计学差异（ｐ＞０．０５），但是ＥＬＩＳＡ检测抗体有１９．０％的假阳性。研究表明，ＰＣＲ是一种特异、敏感、快速诊断结核病的方法。     

PCR与单克隆抗体ELISA夹心法快速诊断结核病的研究

应用聚合酶链反应（ＰＣＲ）技术建立的扩增结核杆菌复合体特异重复序列ＩＳ９８６基因的方法和用单克隆抗体ＴＢ１５－Ｃ３经ＥＬＩＳＡ夹心法检测结核杆菌特异性抗原决定簇，对１０种抗酸杆菌，２种普通菌进行了检测。ＰＣＲ仅对结核杆菌复合体扩增出２４５ｂｐ特异性条带，ＥＬＩＳＡ检测除人型结核杆菌、ＢＣＧ阳性外，还与鸟型及瘰疬分枝杆菌反应阳性。ＰＣＲ检测人型结核杆菌的敏感性为１ｐｇ，相当于１３个左右细菌，ＥＬＩＳ     

母婴弓形体感染的临床研究

应用酶联免疫吸附法（ＥＬＩＳＡ）和聚合酶链反应法（ＰＣＲ）联合对４０６份晚孕妇女静脉血及３２４份新生儿脐带血的弓形体ＩｇＧ、ＩｇＭ、ＤＮＡ片段进行检测，结果表明孕妇弓形体感染率为１５．８％，新生儿感染率为５．９％。证明ＰＣＲ法阳性率高于ＥＬＩＳＡ法测ＩｇＭ（Ｐ＜０．０５）。提出在条件许可医院ＰＣＲ法可代替ＥＬＩＳＡ法而做为治疗指标。对母婴弓形体ＩｇＭ或ＰＣＲ阳性者采用螺旋霉素或磺胺嘧啶、乙胺嘧啶、叶酸等联合治疗，并长期随访患者，以此减少弓形体对母婴的危害。达到优生优育的目的     

DNA复制错误阳性大肠癌的临床病理特点

目的探讨ＤＮＡ复制错误（ＲＥＲ）与大肠癌发生发展的关系。方法采用银染ＰＣＲ单链构象多态性（ｓｉｎｇｌｅｓｔｒａｎｄｃｏｎｆｏｒｍａｔｉｏｎｐｏｌｙｍｏｒｐｈｉｓｍ，ＳＳＣＰ）和ＰＣＲ－变性聚丙烯酰胺凝胶（ＰＡＧ）技术检测６０例大肠癌及其相应正常组织的第２、５、１７号染色体的４个位点的微小卫星ＤＮＡ不稳定性（ｍｉｃｒｏｓａｔｅｌｉｔｅｉｎｓｔａｂｉｔｙ，ＭＳＩ），有至少２个位点的ＭＳＩ则诊断为ＲＥＲ阳性。结果６０例大肠癌中，１９例为ＲＥＲ阳性（３１７％）。结合家族史，根据Ａｍｓｔｅｒｄａｎ标准，６０例病例中有４例被诊断为遗传性非息肉病性大肠癌（ｈｅｒｅｄｉｔａｒｙｎｏｎｐｏｌｙｓｉｓｃｏｌｏｒｅｃｔａｌｃａｎｃｅｒ，ＨＮＰＣＣ）。ＲＥＲ阳性率在ＨＮＰＣＣ中为３／４例与散发性大肠癌的２８５％比差异有显著性（Ｐ＜００５）。ＲＥＲ阳性大肠癌与ＲＥＲ阴性大肠癌比较大多为分化不良型腺癌（Ｐ＜００１），多位于右半结肠（Ｐ＜００５），有家族史（Ｐ＜００５），ＤｕｃｋｅｓＡ、Ｂ期较ＤｕｃｋｅｓＣ、Ｄ期患者所占比例高（Ｐ＜０．０５）。结论ＲＥＲ阳性大肠癌与ＲＥＲ阴性大肠癌有不同的生物学特性。     

用Dot－ELISA检测孕妇巨细胞病毒感染

建立了快速检测孕妇血清内的巨细胞病毒（ＨＣＭＶ）ＩｇＭ、ＩｇＡ的ＤＯｔ－ＥＬＩＳＡ技术，用该法检测１２０例孕妇血清，ＨＣＭＶ－ＩｇＭ、ＩｇＡ的阳性率分别为１２．５０％和１４．１７％；总检出率为２１．６７％，显著高于对照组（Ｐ＜０．０５）。与ＥＬＩＳＡ相比，灵敏性为９６．３０％，特异性１００％。两者对ＨＣＭＶ－ＩｇＭ、ＩｇＡ的检出率无显著性差别（Ｐ＞０．０５），但较ＥＬＩＳＡ更为快速，简便，经济，适于推广普及。     

PCR技术和血清抗体检测妊娠期巨细胞病毒感染价值的比较

将ＰＣＲ技术检测孕妇血白细胞中ＨＣＭＶ－ＤＮＡ和ＥＬＩＳＡ检测孕妇血清中ＨＣＭＶ特生ＩｇＭ和ＩｇＧ进行对比研究。结果：ＤＮＡＰＣＲ阳性率为４２．００％，与ＩｇＭ阳性率２５．５８％比较，Ｐ〈０．０５，与ＩｇＧ阳性率６２．７９％比较，Ｐ〈０．０１，说明ＤＮＡＰＣＲ较ＩｇＭ检测敏感，但不如ＩｇＧ。结论：ＤＮＡＰＣＲ更适用于论断先天性ＨＣＭＶ感染和一部分检测不出ＩｇＭ的ＣＭＶ复发感染者，对妊娠期ＣＭＶ感染     

组合单克隆抗体夹心斑点-ELISA检测日本血吸虫循环抗原的研究

本研究将抗不同表位单抗组合，首次用于夹心斑点-ＥＬＩＳＡ检测血吸由感染。对慢性日本血吸虫病患者的阳性检出率为８５％，１２例急性患者均阳性，１０例晚血病人４例阳性，非疫区正常人的假阳性率为２．２％。用单盲法检测的-组血清其符合率亦较满意。在基本消灭血吸虫病地区检测了１１００人，用夹心斑点-ＥＬＩＳＡ测抗原，阳性率为６．５％，以ＩＨＡ测抗体阳性率为２５．２％。在低年龄组人群，斑点ＥＬＩＳＡ与ＣＯＰＴ符合率为８８．９％，明显高于ＩＨＡ与ＣＯＰＴ的符合率（３５．７％），Ｐ＜０．０５。动物实验中，组合单抗夹心班点ＥＬＩＳＡ对低感染兔的阳性检出率高于单个单抗直接斑点-ＥＬＩＳＡ。     

新生儿硬肿症患儿尿视黄醇结合蛋白改变的临床意义

为了解新生儿患儿肾小管功能变化，应用ＥＬＩＳＡ法检测３６ 例新生儿硬肿症患儿尿视黄醇结合蛋白（ＲＢＰ）。结果显示硬肿症患儿尿ＲＢＰ明显高于对照组（ Ｐ＜ ０．０１） ，中、重度硬肿症组均高于对照组（ Ｐ＜０ ．０５ ，Ｐ＜ ０．０１） 。而轻度硬肿症组与对照组的差异无显著性（ Ｐ＞ ０ ．０５） ，提示中、重度新生儿硬肿症患儿存在有肾小管功能的损害。     

急性早幼粒细胞白血病患者诱导分化治疗前后血清IL—2受体的…

ＥＬＩＳＡ法检测２７例急性早幼粒细胞白血病患者（ＡＰＬ）血清白细胞介素２受体（ｓＩＬ－２Ｒ）水平。结果表明ＡＰＬ患者血清ｓＩＬ－２Ｒ高于正常对照（Ｐ〈０．０１），经维甲酸治疗后逐渐降低，但治疗中期患者血清ｓＩＬ－２Ｒ仍高于正常和治疗后（缓解期）患者血清ｓＩＬ－２Ｒ（Ｐ〈０．０５）。治疗后血清ｓＩＬ－２Ｒ虽略高于正常对照。却已恢复正常范围（Ｐ〈０．０５）。同时发现治疗中ＷＢＣ却明显升高，治疗后又下降     

bcl-2、p53蛋白及PCNA表达与横纹肌肉瘤临床病理相关性研究

目的：研究横纹肌肉瘤（ＲＭＳ）中ｂｃｌ－２、ｐ５３、ＰＣＮＡ表达与其临床病理的相关性。方法：对５０例（随访４１例）横纹肌肉瘤进行免疫组化ＡＢＣ法标记。结果：ｂｃｌ－２、ｐ５３基因蛋白和ＰＣＮＡ，发现ｂｃｌ－２、ｐ５３、ＰＣＮＡ阳性表达率分别为２８％、７２％、７０％，其阳性表达与年龄、性别及不同组织类型的ＲＭＳ无关（Ｐ＞０．０５）。但与分化程度有关，ｐ５３、ＰＣＮＡ在低分化ＲＭＳ阳性率分别为８５％、９５％，显著高于高分化ＲＭＳ４２．８％和１４．３％（Ｐ＜０．０５），随访存活１年以内的ｐ５３、ＰＣＮＡ阳性率均为８６．７％，亦明显高于存活超过３年以上的阳性率３３．３％和４１．７％（Ｐ＜０．０５）。而ｂｃｌ－２在低分化ＲＭＳ阳性２０％显著低于高分化７１．４％（Ｐ＜０．０５），随访存活１年以内的阳性率１３．４％明显低于存活超过３年以上的４１．４％（Ｐ＜０．０５）。ｐ５３与ｂｃｌ－２阳性表达呈明显负相关，ｐ５３阳性率越高，而ｂｃｌ－２阳性率越低。结论：ＰＣＮＡ、ｐ５３、ｂｃｌ－２蛋白表达能比较准确地反映ＲＭＳ的生物学特性，ｐ５３、ｂｃｌ－２可作为肿瘤预后显著相关的有效指标。     

用PCR技术对淋巴结核进行病原学检查的初步研究

By using polymerase chain reaction (PCR) technique in amplifying a reparative DNA sequence specific for Mycobacterium tuberculosis, the pathogenic bacterium in paraffin-embedded tissue was identified. A 123-base-pair fragment, specific PCR product, was obtained from all 12 cases of tuberculous lymphadenitis and 4 out of 10 cases in which tuberculosis was suspected. In the 4 suspected cases, 3 cases showed good results when treated by anti-tuberculosis therapy. The remaining case was lost from follow-up. Comparing acid-fast stain and PCR results, we confirm that PCR method is more powerful and more sensitive than acid-fast stain in the etiological diagnosis of tuberculous lymphadenitis.     

Rapid and specific detection of Mycobacterium tuberculosis by using the Smart Cycler instrument and a specific fluorogenic probe

A procedure using the Smart Cycler instrument and a fluorescence quencher (FQ) probe for the specific identification of Mycobacterium tuberculosis complex (MTB) was used to detect organisms in 366 acid-fast bacillus smear-positive respiratory specimens. It was compared to culture and the AMPLICOR M. tuberculosis PCR test. MTB was isolated from 198 of these samples. The FQ PCR assay was sensitive (197 of 198, 99.5%) and specific (165 of 168, 98.2%); no significant difference was observed between the two PCR protocols. After DNA extraction, a final result was available within 1.5 h with the real-time PCR protocol.     

Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplified procedure.

A repetitive sequence of Mycobacterium tuberculosis DNA was amplified by polymerase chain reaction (PCR), from sputum samples, for the diagnosis of pulmonary tuberculosis. The method of heating the sample in a boiling water bath to break down the bacterial cell wall and to release the DNA was compared with that of enzymatic lysis of bacteria and then phenol-chloroform extraction of DNA. Heating the sample was the better method with a sensitivity of approximately 10 microorganisms. A total of 78 sputum specimens prepared by heating were examined by PCR, and the results were compared with the results of acid-fast stained smears, cultures, and clinical data. M. tuberculosis was detected by PCR in all smear- and culture-positive and smear-negative, culture-positive cases. Additionally, PCR was capable of detecting four of nine cases which were smear and culture negative but clinically suspected of tuberculosis. DNA amplification by PCR is a sensitive and specific method for the diagnosis of tuberculosis, and with this simplified DNA isolation procedure it can be used in routine clinical practice.     

Multiprimer PCR system for differential identification of mycobacteria in clinical samples.

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obtained, using reference mycobacterial strains and typed clinical isolates, show that the multiprimer PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay.     

Detection of mycobacterial nucleic acids by polymerase chain reaction in fixed tissue specimens of patients with human immunodeficiency virus infection. Swiss HIV Cohort Study.

A polymerase chain reaction (PCR) method, which amplifies a fragment of the 16S ribosomal RNA (rRNA) gene present in all mycobacterial species, was developed and tested on 84 formalin-fixed paraffin-embedded tissue specimens from 51 patients with human immunodeficiency (HIV) infection. The PCR products were characterized either by sequencing or by hybridization with nonradioactive oligonucleotide probes specific for Mycobacterium tuberculosis complex, M. avium, or M. genavense. Sequencing was successful for 26 samples compared with the 45 samples for probe hybridization. The sensitivity of DNA amplification compared with microscopic examination was 79.5%. A mixed infection was detected with M. genavense for only one patient who was infected with M. tuberculosis complex. In the group of 22 control patients, where no diagnosis of mycobacterial infection was made during life and no acid-fast bacteria were seen during the autopsy, four samples of one patient were positive by hybridization with the M. tuberculosis probe. This patient had a clinical history compatible with tuberculosis. This PCR method may be a powerful tool for the precise diagnosis of mycobacterial infections from histopathologic material, provided that several sections from the same specimen block are tested.     

Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR.

Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.     

Use of the real-time PCR assay in conjunction with MagNA Pure for the detection of mycobacterial DNA from fixed specimens.

Tuberculosis in immunocompromised patients is often caused by Mycobacterial species other than Mycobacterium tuberculosis. Thus, detection of and differentiation between M. tuberculosis and nontuberculosis species is necessary for diagnosis of disease in these patients. Furthermore, when tissue changes show granulomatous inflammation, quick confirmation testing for mycobacterial infection is needed for conclusive diagnosis. The aim of this study was to validate the utility of a real-time polymerase chain reaction (PCR) assay in conjunction with the MagNA Pure LC automated extraction system for the detection of mycobacterial DNA from formalin-fixed, paraffin-embedded specimens. A total of 46 archived, paraffin-embedded, fixed specimens showing granulomatous inflammation were studied for mycobacterial infection by real-time PCR. Bacterial DNA was extracted and isolated using the MagNA Pure extraction system. Real-time PCR was performed on the LightCycler using the Artus Real Art Mycob Diff ASR kit from Qiagen. Thirteen of the 46 patient specimens were positive for mycobacterial infection by acid-fast bacilli (AFB) stain. Of the13 reported positive by AFB stain, 12 where positive by real-time PCR. All 13 specimens reported positive by AFB were sent for culture confirmation. Eleven of 13 were returned positive by culture. Specimens reported as negative by culture and positive by real-time PCR were confirmed positive by a second PCR method from another reference laboratory. We believe that these studies are beneficial in the differential diagnosis of mycobacterial infection from fixed tissue specimens where tuberculosis might not have been clinically initially suspected and when specimens are not suitable for microbiologic examination.     

Rapid and specific detection of Mycobacterium tuberculosis from acid-fast bacillus smear-positive respiratory specimens and BacT/ALERT MP culture bottles by using fluorogenic probes and real-time PCR

A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of Mycobacterium tuberculosis complex (MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC PCR assay was directed at the amplification of the internal transcribed spacer region of the Mycobacterium genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The results from the respiratory specimens were compared to those from the Amplicor M. tuberculosis PCR test. Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms; both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample available for testing.     

Use of PCR in routine diagnosis of treated and untreated pulmonary tuberculosis.

AIMS--To assess the routine use of a polymerase chain reaction (PCR) assay for the direct detection of Mycobacterium tuberculosis in expectorated sputum specimens. METHODS--A pair of primers (20-mer) were designed to amplify the 38 kilodalton protein of M tuberculosis. The specificity of the assay was evaluated in 31 M tuberculosis strains, 15 atypical mycobacterium species, and several commensal bacteria of the upper respiratory tract. The assay was subsequently applied to 519 sputum specimens from 85 inpatients of a chest hospital in Hong Kong. RESULTS--An amplified product of 239 base pairs was found in all M tuberculosis strains, standard strains of M bovis, and M africanum but not in the other bacterial strains tested. For the 51 patients with pulmonary radiographic lesions, the diagnosis of pulmonary tuberculosis was subsequently confirmed by both culture and PCR in 41 of them. Five patients who were treated before admission were positive by PCR alone. All but one patient in the control group (patients with acute exacerbation of chronic obstructive airway diseases) or those with atypical mycobacterial diseases were PCR negative. The PCR remained positive after four weeks of anti-tuberculosis treatment in 29 patients, 16 of whom had become culture negative. CONCLUSION--This PCR assay is a useful technique for the diagnosis of untreated and recently treated cases of pulmonary tuberculosis.     

Comparison of in-house PCR with conventional techniques and Cobas Amplicor M. tuberculosis kit for detection of Mycobacterium tuberculosis

PURPOSE: Polymerase chain reaction (PCR) assay, introduced as a fast and sensitive diagnostic method, is useful in detecting Mycobacterium tuberculosis. The purpose of this study was to evaluate the usefulness of in-house PCR assay in the detection of Mycobacterium tuberculosis by comparing PCR results with conventional diagnostic techniques and Cobas Amplicor M. tuberculosis kit. MATERIALS AND METHODS: We retrospectively assessed the diagnostic yield of in-house PCR method employed for the amplification IS6110 sequences in 2,973 specimens. We also compared in-house PCR with Cobas Amplicor M. tuberculosis kit in 120 specimens collected from June to July 2006. Routine acid-fast stain (AFS) and culture assay were also performed and analyzed. RESULTS: Of 2,973 cases, 2,832 cases (95.3%) showed consistent results between in house PCR, AFS and culture methods, whereas 141 (4.7%) displayed inconsistent results. The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 77.5%, 99.7%, 95.5%, and 98.0%, respectively for PCR; 49.2%, 100%, 100%, and 95.7%, respectively, for AFS method; and 80.7%, 100%, 100%, and 98.3%, respectively, for culture assay. Consistent results between PCR and Cobas Amplicor M. tuberculosis kit were shown in 109 cases (90.8%). The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 81.3%, 98.9%, 96.3%, and 93.5% respectively for PCR and 71.9%, 100%, 100%, and 90.7%, respectively, for Cobas Amplicor kit. CONCLUSION: In-house PCR and Cobas Amplicor kit show high sensitivity and specificity, and are reliable tests in the diagnosis of tuberculosis.