分子信标用于结核分枝杆菌的均相荧光PCR检测

1996年Ｔｙａｇｉ等〔1〕提出的分子信标是一种具有颈环构型的分子探针 ,用于ＰＣＲ扩增产物均相测定的原理是 ,在退火阶段 ,分子信标与生成的靶序列结合发出荧光 ,在延伸阶段 ,则脱离靶序列而不干扰扩增 ,随着循环次数的增加 ,与模板结合的分子信标的量亦增加 ,最终的荧光强度便与模板量成正相关。我们在原来“分子信标”原理基础上 ,对其设计思想进行了重要改进 ,合成出效率更高的分子信标 ,并自行研制了简便适用的荧光测定装置。本文报告用于结核分枝杆菌的检测结果。材料和方法菌株 :分枝杆菌属包括堪萨斯分枝杆菌、瘰疬分枝杆菌、耻垢…     

EDTA修复肉芽肿组织中荧光定量PCR检测结核杆菌的应用

目的观察EDTA热修复炎性肉芽肿组织石蜡切片,能否提高荧光定量PCR结核/非结核分枝杆菌的检出率。方法对125例炎性肉芽肿组织石蜡切片结核/非结核分枝杆菌同时进行常规荧光定量PCR检测和EDTA热修复后荧光定量PCR检测。结果常规荧光定量PCR检测检出分枝杆菌75例(60%)阳性,其中结核杆菌74例(59.2%),非结核分枝杆菌1例(0.8%),阳性病例平均阳性基因拷贝数6.35×105/ml/例;EDTA修复后荧光定量PCR检测检出分枝杆菌88例(70.4%),其中结核杆菌83例(66.4%),非结核分枝杆菌5例(4%),阳性病例平均阳性基因拷贝数7.36×106/ml/例。两组间差异均有统计学意义(P0.05,P0.01)。结论 EDTA热修复后荧光定量PCR检测可以大幅度提高炎性肉芽肿组织石蜡切片的结核/非结核分枝杆菌的阳性检出率。     

应用荧光定量PCR法检测病理组织中幽门螺旋杆菌感染

目的探讨荧光定量PCR方法检测病理组织中幽门螺旋杆菌（Hp）的效果,为临床Hp感染诊断提供快速有效的检测方法。方法应用荧光定量PCR法及苯胺蓝染色法分别对106例临床送检胃窦黏膜组织标本进行幽门螺旋杆菌检测,对检测结果进行对比分析。结果在106例胃窦标本中,荧光定量PCR法检测阳性例数68例,阳性率为64．2％,苯胺蓝染色法检测阳性例数46例,阳性率为43．4％。两种方法检测阳性率比较有显著性差异（P〈0．05）。结论荧光定量PCR技术用于检测病理组织中幽门螺旋杆菌,具有灵敏度高、特异性强的特点,对确诊胃镜活检标本中幽门螺旋杆菌感染具有重要应用价值。     

荧光PCR法检测石蜡标本中结核分枝杆菌的流程改进

<正>结核分枝杆菌的检测对于有肉芽肿性炎特征组织病变的病理诊断具有重要的参考意义。目前,病理科常用的检测方法有基于细菌学的抗酸染色、金胺O荧光染色,基于分子病理学的PCR、Xpert MTB/RIF检测^[1-2]等。其中荧光PCR技术具有敏感性高、特异性好、速度快,且价格实惠的特点,被广泛应用于大中型医院病理科福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded,FFPE）组织的结核分枝杆菌的检测中^[3-6]。由于石蜡组织标本中的病菌分布不稳定,     

基于PCR的分子生物学技术在检测非结核分枝杆菌中的进展

非结核分枝杆菌（NTM）病是指人体感染NTM,并引起相关组织、脏器的病变。传统检测方法对该病的诊断较为困难。近年来随着分子生物学技术的发展,特别是聚合酶链式反应（PCR）技术的出现,涌现出新的一批基于PCR技术对NTM病进行快速检测的产品。文章以PCR技术为切入点,对限制性片段长度多态性PCR技术、分子线性探针技术、荧光定量PCR熔解曲线法、PCR-基因测序法、巢式PCR、多重PCR、荧光定量PCR技术和数字PCR的技术原理及在检测NTM中的研究进展进行回顾和分析,这些技术具有快速、操作简便、鉴定准确等优势。应用前景,相信在未来将更加完善这些鉴定方法。     

蛋白芯片检测结核分枝杆菌对肺结核病的临床诊断价值

肺结核病是一种由结核分枝杆菌引起的慢性传染病,是我国重点控制的重大疾病之一。目前结核病的诊断方法有：痰涂片、结核菌培养、结核菌基因检测、胸部X线和CT等,但这些方法存在特异性差或需要时间长、敏感性低等缺点。因此,敏感、特异、快速的检测方法对肺结核病的诊断与鉴别十分重要。     

噬菌体法检测结核分枝杆菌的临床应用

研究噬菌体扩增试验对结核分枝杆菌快速鉴定的意义.应用结核分枝杆菌噬菌斑技术快速检测结核分枝杆菌及肺结核患者痰标本.结果显示:结核分枝杆菌H37 Rv、噬菌体扩增试验均为阳性;20株结核分枝杆菌临床分离株本法检测结果全部阳性;20份涂阳肺结核患者(用药时间在一周之内的)痰标本,本法阳性19份(95％);20份涂阴(应用抗...     

等位基因特异多重PCR快速检测结核分枝杆菌异烟肼耐药性研究

了解北京地区异烟肼（INI-I）耐药结核分枝杆菌（MTB）的分子特点,评价等位基因特异多重PCR在INH耐药性快速检测中的应用价值。选取102株北京地区MTB临床分离株,首先分别采用PCR—RFLP和反向线性杂交技术（RLB）检测耐药相关基因katG和inhA突变,然后运用等位基因特异多重PCR方法同时检测katG和inhA基因突变,并与PCR—RFLP和RLB分别检测的结果进行比较。采用等位基因特异多重PCR检测发现,INH耐药株中60．3％（35／58）存在katG315突变,13．8％（8／58）发生了inhA突变,两者同时突变率为6．9％（4／58）,INH敏感株中没有发现katG315突变,而inhA突变率为18．2％（8／44）。上述检测结果与PCR-RFLP和RLB分别检测的结果完全一致,且等于两种方法结果之和,提示等位基因特异多重PCR可完全取代PCR—RFLP和RLB。北京地区INH耐药菌株以katG315 AGC→ACC突变为主;联合检测katG315和inhA基因可提高INH耐药株的检出率。等位基因特异多重PCR操作简便,结果易于判断,可作为临床MTB菌株中INH耐药性快速检测的辅助手段。     

抗酸染色与即时荧光定量PCR法检测结核分枝杆菌在辅助诊断结核病中的作用比较北大核心CSCD

在我国结核病被列为重大传染病之一,是严重危害国民健康的呼吸道传染病,根据世界卫生组织（WHO）的统计,我国结核病患病人数居世界第2位,是全球22个结核病流行严重的国家之一。结核病的早发现、早诊断、早治疗在防控工作中尤为重要。     

PCR-膜芯片检测石蜡包埋组织中结核杆菌耐药基因突变

目的探讨PCR-膜芯片技术检测石蜡包埋组织中结核杆菌耐药基因突变的可行性及其在临床病理中的应用价值。方法采集100例石蜡包埋的肺或淋巴结组织标本(临床诊断为结核患者80例,非结核患者20例),TB膜芯片检测标本中的结核杆菌IS6110基因,阳性者利用12对特异性引物进行多重PCR,扩增产物与含有59个探针的耐药-TB膜芯片反向点杂交检测结核杆菌耐药基因突变并测序验证。结果 TB膜芯片检测石蜡包埋组织标本中的结核杆菌IS6110基因,以临床诊断为标准时,灵敏度为52.5%(42/80),特异度为100%(20/20);以抗酸染色结果为标准时,灵敏度为90.5%(19/21),特异度为61.0%(36/59)。耐药-TB膜芯片检测石蜡包埋组织结核杆菌耐药基因突变,在42例结核杆菌IS6110基因阳性的标本中,检出INH基因突变2例,RFP或SM基因突变各1例,INH、RFP和EMB基因同时突变1例,与测序结果基本符合,提示上述5例可能为耐药结核病。结论应用PCR-膜芯片技术可提高石蜡包埋组织中结核杆菌基因的检出率,耐药-TB膜芯片检测结果可为耐药结核病的病理学诊断提供有价值的参考。     

结核杆菌HSP65 DNA疫苗的初步研究

目的 构建以结核分枝杆菌热休克蛋白65000u基因为基础的核酸疫苗。方法 采用聚合酶链反应从结核杆菌H37Rv株基因中，扩增出HSP65的编码基因，经限制性核酸内切酶消化后，插入真核表达载体pcDNA3.1(－）的相应酶切位点，并将此重组质粒免疫动物。结果 重组质粒的插入基因经序列测定证实为结核分枝杆菌HSP65。DNA免疫小鼠体内产生特异性抗体。结论 以HSP65编码基因为基础的真核表达载体构建成功，并能引起特异性动物免疫反应，为进一步研究其在结核病防治中的作用奠定了基础。     

结核分枝杆菌Ag85B基因疫苗免疫保护作用的初步研究

目的:研究编码结核分枝杆菌分泌蛋白Ag85B的基因疫苗pTB30m和pTB30s对免疫动物的保护作用。方法:以基因疫苗pTB30m和pTB30s肌注免疫BALB/c小鼠。免疫完成6 wk后,用5×105 CFU的MTB H37Rv毒株经小鼠尾静脉攻击感染。同时用尼龙毛柱分离基因免疫BALB/c小鼠的T细胞,并以5×106 T细胞/只小鼠过继免疫正常BALB/c小鼠,立即用105 CFU的MTB毒株经小鼠尾静脉攻击感染。4 wk后分别计数脾脏中的细菌负荷。结果:与生理盐水对照组相比较,pTB30m及pTB30s质粒免疫组BALB/c小鼠脾脏中的细菌负荷均减少,分别为0.645(log10 CFU,P<0.01)和0.839(log10CFU,P<0.001);而空质粒对照组小鼠脾脏中的细菌负荷减少较少。经质粒pTB30m和pTB30s免疫的BALB/c小鼠的T细胞,过继免疫的正常BALB/c小鼠,对攻击感染的MTB H37Rv毒株在脾脏中的增殖具有部分抑制作用。结论:pTB30s免疫的BALB/c小鼠,对MTB H37 Rv毒株攻击的保护作用优于pTB30m质粒免疫,有望进一步用于结核病的防治研究。     

Evaluation of two tuberculosis PCR assays for routine use in a clinical setting of low population and low tuberculosis prevalence

Today, there are numerous different molecular diagnostic assays for the detection of tuberculosis (TB), allowing the optimization of rapid detection of TB according to the clinical need. In this study, two high‐throughput TB PCR assays with combined antimicrobial resistance detection, Anyplex? II MTB/MDR (Seegene) and RealTime MTB + RealTime MTB RIF/INH Resistance (Abbott Molecular), were evaluated for routine use in a clinical setting of low population and low TB prevalence in Finland. The RealTime MTB assay was 100% concordant (22/22 positive, n = 169) with the reference methods (culture and Xpert MTB/RIF PCR assay, Cepheid). However, with a limitation of four separate PCR cycles per kit, the routine use in a low TB‐prevalence setting would easily lead to wasting most of the RIF/INH Resistance reagents. The Anyplex? II MTB/MDR assay usability was more adaptive to suit the clinical setting but the assay sensitivity was considerably lower (86%, 19/22 positive, n = 76) being closer to the sensitivity of smear microscopy. The findings of this study suggest that the evaluated high‐throughput MTB/MDR assays are evidently suboptimal for routine use in a low population, low TB‐prevalence setting. In addition, neither of the two assays covers non‐tuberculous mycobacteria and could therefore not fully replace acid‐fast staining as the initial screening method.     

Evaluation of the GenoType MTBDR assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates

Detection of drug resistance plays a critical role in tuberculosis treatment. The aim of this study was to evaluate the performance of GenoType Mycobacteria Drug Resistance (MTBDR) assay (Hain Lifescience, Germany) and to compare it with radiometric BACTEC 460 TB system (Becton Dickinson, USA) for the detection of rifampicin (RIF) and isoniazid (INH) resistance in 84 Mycobacterium tuberculosis complex (MTBC) isolates. RIF resistance was identified in 6 of 7 (85.7%) isolates and INH resistance was identified in 8 of 14 (57.1%) isolates by the GenoType MTBDR assay. Compared with BACTEC system, the sensitivity, specificity, positive predictive value and negative predictive values were 85.7%, 98.7%, 85.7% and 98.7% for RIF resistance; and 57.1%, 100%, 100% and 92.1% for INH resistance, respectively. GenoType MTBDR assay is reliable when tested specimen is resistant to the tested drugs. Although test was more successful in the detection of RIF resistance, it exhibited low sensitivity for the detection of INH resistance.     

Fluorescent quantitative PCR of Mycobacterium tuberculosis for differentiating intestinal tuberculosis from Crohn's disease

Intestinal tuberculosis (ITB) and Crohn''s disease (CD) are granulomatousdisorders with similar clinical manifestations and pathological features thatare often difficult to differentiate. This study evaluated the value offluorescent quantitative polymerase chain reaction (FQ-PCR) forMycobacterium tuberculosis (MTB) in fecal samples andbiopsy specimens to differentiate ITB from CD. From June 2010 to March 2013, 86consecutive patients (38 females and 48 males, median age 31.3 years) withprovisional diagnoses of ITB and CD were recruited for the study. The patients''clinical, endoscopic, and histological features were monitored until the finaldefinite diagnoses were made. DNA was extracted from 250 mg fecal samples andbiopsy tissues from each patient. The extracted DNA was amplified using FQ-PCRfor the specific MTB sequence. A total of 29 ITB cases and 36 CD cases wereincluded in the analysis. Perianal disease and longitudinal ulcers weresignificantly more common in the CD patients (P<0.05), whereas night sweats,ascites, and circumferential ulcers were significantly more common in the ITBpatients (P<0.05). Fecal FQ-PCR for MTB was positive in 24 (82.8%) ITBpatients and 3 (8.3%) CD patients. Tissue PCR was positive for MTB in 16 (55.2%)ITB patients and 2 (5.6%) CD patients. Compared with tissue FQ-PCR, fecal FQ-PCRwas more sensitive (X²=5.16, P=0.02). We conclude that FQ-PCR for MTBon fecal and tissue samples is a valuable assay for differentiating ITB from CD,and fecal FQ-PCR has greater sensitivity for ITB than tissue FQ-PCR.     

Evaluation of a nested PCR targeting IS6110 of Mycobacterium tuberculosis for detection of the organism in the leukocyte fraction of blood samples

Purpose: Tuberculosis poses a serious health problem in resource-poor settings such as India. Polymerase chain reaction (PCR) is presently seen as a promising alternative to conventional smear microscopy and culture techniques. Undiagnosed fever is a condition where the aetiology could include tuberculosis in a significant percentage. This paper evaluates a nested PCR (nPCR) using Hotstar Taq for the detection of M. tuberculosis in patients with febrile illness using insertion element, IS6110 as a target. Material and Methods: A total of 355 samples (301 HIV status unknown and 54 HIV seropositives) from patients primarily with febrile illness were tested for the presence of M. tuberculosis. Blood culture was done in a commercial automated blood culture system and nPCR in DNA extracts from buffy coat samples. Hotstar Taq polymerase was used to enhance the sensitivity of nPCR and the lower limit of detection was determined by using cloned plasmid. Results: Among the patients tested, 2% were positive by automated culture system and 6.8% of patients were positive by nPCR. Majority of the positives were from HIV seropositive individuals. The sensitivity of the nPCR was 100% and the specificity was 95.1%. The lower limit of detection was less than 1 genome copy per microlitre. Among the nPCR positives, patients from rural community were significantly higher than from the peri-urban community. Conclusions: The nPCR had a high sensitivity and specificity on buffy coat samples using Hotstar Taq polymerase in the reaction mix. Thus the technique is a valuable tool in the diagnosis of tuberculosis.     

Characterization of Mycobacterium tuberculosis isolated from cancer patients with suspected tuberculosis infection in Egypt: identification,prevalence, risk factors and resistance pattern

Data are sparse on Mycobacterium tuberculosis infection among patients with cancer in Egypt. We sought to detect the presence of tuberculosis (TB) disease among patients with malignant conditions and suspected TB and to study the main risk factors. Also, we compared different diagnostic procedures and detected the antimicrobial susceptibility of M. tuberculosis isolates against rifampin and isoniazid. One hundred patients were included in this study, all of them had malignant conditions and were suspected by the clinicians of having TB. Identification of M. tuberculosis in different specimens was performed by smear microscopy, followed by Lowenstein–Jensen medium and Mycobacterium growth indicator tube (MGIT) cultures and artus^® real-time PCR. In addition, an indirect MGIT anti-TB susceptibility test was carried out against rifampin and isoniazid. A total of 76% of studied cases were found to be TB positive. The frequencies of TB-positive cases in the bronchogenic, haematological and solid tumour malignancy groups were 21%, 25% and 30%, respectively. Significant differences between pulmonary and extrapulmonary TB in different malignancy groups were recorded. Real-time PCR showed the highest overall diagnostic efficiency. Multidrug-resistance of M. tuberculosis to both rifampin and isoniazid was detected in 28.6% of examined isolates. Infection in cancer patients with TB was significantly more often recorded among elderly patients and those suffering from poverty. Pulmonary TB is more common than extrapulmonary TB in patients with malignancy. Real-time PCR is the most accurate and rapid method for TB diagnosis. MGIT-rifampin resistance may be used as a reliable marker for detection of multidrug-resistant TB. Diagnosis and instituting treatment course for active or latent TB infection are crucial before starting anticancer therapy.