猪圆环病毒3型TB GreenⅡ实时荧光定量PCR检测方法的建立和应用

为建立猪圆环病毒3型(Porcine circovirus 3,PCV3)快速、灵敏且特异的检测方法,针对PCV3全基因组的保守区域设计特异性引物,构建标准质粒,通过优化反应体系和反应程序,建立基于TB GreenⅡ的实时荧光定量PCR(qPCR)方法,并对其特异性、敏感性、重复性和可行性进行验证。结果显示,建立的TB GreenⅡqPCR检测方法特异性良好,在4.74×10^(2)~4.74×10^(7)拷贝/μL的标准质粒间具有良好的线性关系(R^(2)=0.999)。建立检测方法的最低检出限为10拷贝/μL,与猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV2)无交叉反应,组内变异系数为0.33%~0.62%,组间变异系数为0.40%~0.73%。对收集的132份临床样品进行检测,PCV3 TB GreenⅡqPCR方法检出率为9.10%(12/132),高于PCV3常规PCR方法的检出率(4.55%,6/132)。综上,建立的PCV3 TB GreenⅡqPCR检测方法具有良好的敏感性、特异性和重复性,可用于临床样品中PCV3感染的诊断、流行病学监测和实验室研究等。

刮取涂片PCR检测TB方法及应用的探讨

刮取涂片ＰＣＲ检测ＴＢ方法及应用的探讨张素娟，董敬朋，张文丽，陆药丹，赵彤（广州第一军医大学病理学教研室，广州５１０５１５）本室结合临床情况，并参照ｓｕｋｐａｎｉｃ－ｈｍａｎｔ等人关于从存档切片上刮取组织进行ＩｇＨ链基因重排的ＰＣＲ扩增，将未查到癌细...

TB-PCR与胸膜活检在结核性渗出性胸膜炎中的应用价值

目的研究结核分枝杆菌荧光定量聚合酶链反应(TB-PCR)检测技术及胸膜活检在诊断结核性渗出性胸膜炎的临床价值。方法 282例确诊为结核性渗出性胸膜炎患者行胸腔穿刺抽液术,胸液送检TB-PCR,同时行针刺胸膜活检,胸膜活检组织标本送检病理检查,比较两种检测方法的阳性率。结果 TB-PCR检测阳性率55.3%,明显高于胸膜活检阳性率37.9%,差异有高度统计学意义(P<0.01),聚合酶链式反应检测明显优于胸膜活检。两种方法联合检出阳性率为64.2%。结论 TB-PCR是目前针对结核分枝杆菌检测阳性率较高的方法;联合胸膜活检,可提高对结核性渗出性胸膜炎的早期诊断。

鹅圆环病毒TB Green实时荧光定量PCR方法的建立及初步应用

鹅圆环病毒主要感染宿主的淋巴细胞,导致其免疫功能下降,造成继发感染和共感染。为建立鹅圆环病毒分子检测方法,本研究根据NCBI数据库中GoCV基因特征,设计特异性引物,建立检测GoCV的TB Green实时荧光定量PCR方法。结果显示,建立检测GoCV的TB Green实时荧光定量PCR方法,灵敏度高,最低检测限为54.10拷贝/μL;特异性好,与水禽常见传染病病原均无交叉反应,扩增产物的熔解曲线仅GoCV出现1个特异性单峰,Tm值为(84.46±0.09)℃;重复性好,组内和组间重复试验变异系数分别为0.26%~0.55%和0.23%~0.87%。利用建立的TB Green实时荧光定量PCR方法和常规PCR方法同时对64份临床样品进行GoCV感染的检测,结果显示,常规PCR方法检出阳性样品15份,阳性率为23.44%;TB Green实时荧光定量PCR方法检出阳性样品18份,阳性率28.13%,且15份PCR阳性样品经TB Green实时荧光定量PCR方法检测均为阳性,符合率为100%。本研究为后续开展GoCV感染的分子流行病学调查和致病机理研究提供技术支撑。

均相荧光探针PcR技术在TB检测中的研究

本实验利用均相荧光探针 Pc R技术对结核病患者痰液进行了检测 ,结果说明 ,采用均相荧光测量方式 ,提高了 Pc R检测的特异性与灵敏度 ,同时使实验操作步骤简化 ,杜绝扩增产物污染的途径及人工观察电泳的主观性 ,可适用于各种病原体、点突变等基因诊断 ,使 Pc

Diagnostic Value of TB-IGRA, PPD, TB-DNA-PCR and TB-Ab in Silicosis Complicated with Tuberculosis

Purpose: The purpose of this article is to investigate the clinical value of TB-IGRA (Tuberculosis-Interferon Gamma Release Assay), PPD (Intradermal Terbuculin Test), TB-DNA-PCR (Tuberculosis-Deoxyribonucleic-Polymerase Chain Reaction) and TB-Ab (Tuberculosis-Antibody) in diagnosing silicosis complicated with pulmonary tuberculosis. Methods: 53 cases of suspected silicosis complicated with pulmonary tuberculosis were selected in the time span ranging from February 2017 to May 2019. TB-IGRA test, PPD test, TB-DNA-PCR and TB-Ab detection were performed. The sensitivity, specificity, positive predictive value and negative predictive value were calculated. Results: Silicosis and pulmonary tuberculosis were diagnosed in 11 cases, with an incidence of 20.75%. The positive rates of TB-IGRA, PPD, TB-DNA-PCR and TB-Ab were 66.04%, 30.19%, 5.67% and 26.42%, respectively. The sensitivity was 90.91%, 81.82%, 27.27% and 54.55% respectively. The specificity was 42.86%, 80.95%, 100% and 80.95% respectively. The positive predictive values were 28.57%, 50%, 100% and 42.86% respectively. The negative predictive values were 94.44%, 91.89%, 84% and 87.18%. The positive rate, sensitivity and negative predictive value of TB-IGRA were the highest, while the specificity of TB-DNA-PCR was the highest yet with low positive rate, sensitivity and positive predictive value. Conclusion: The positive rate and sensitivity of TB-IGRA were high, yet with poor specificity, so it was impossible to judge whether the cases belonged to active pulmonary tuberculosis. The combination of PPD and TB-DNA-PCR could improve the sensitivity, specificity and positive predictive value, and the diagnostic accuracy of active pulmonary tuberculosis, which showed satisfactory clinical value.

TB-PCR、Xpert MTB/RIF、T-SPOT释放试验诊断结核感染的临床应用研究

目的:检测TB-PCR、Xpert MTB/RIF、T-SPOT释放试验在结核感染中的诊断及价值评价。方法活动性肺结核组256例,非结核组182例,痰液标本行TB-PCR、Xpert MTB/RIF检测,血液标本行T-SPOT释放试验检测。结果 TB-PCR、Xpert MTB/RIF、T-SPOT检测阳性率分别为31.6%、75.9%和96.5%;检测结果特异性均为100%。且3者之间检查结果差异有统计学意义。结论在诊断结核感染方面, Xpert MTB/RIF检测的应用价值明显高于 TB-PCR检测及Xpert MTB/RIF检测;三者联合应用可提高结核的诊断价值。

Diagnostic Value of TB-IGRA, PPD, TB-DNA-PCR and ADA in Tuberculous Pleural Effusion

Objective: To investigate the clinical diagnostic value of TB-IGRA (Tuberculosis-Interferon Gamma Release Assay), PPD (Intradermal Tuberculin Test), TB-DNA-PCR (Tuberculosis-Deoxyribonucleic-Polymerase Chain Reaction) and ADA(Adenosine Aeaminase) in tuberculous pleural effusion. Methods: 60 patients with tuberculous pleural effusion discharged from our department from January 1, 2018 to December 31, 2019 were selected. Moreover, the TB-IGRA in peripheral blood, PPD test, TB-DNA-PCR and ADA in pleural effusion were detected. Subsequently, the positive rate, negative rate, sensitivity and omission diagnostic rate of TB-IGRA, PPD, TB-DNA-PCR, ADA and combined TB-IGRA were calculated. Results: The positive rate and sensitivity of TB-IGRA, PPD,TB-DNA-PCR, and ADA were 95%, 71.67%, 5% and 86.67% respectively. The omission diagnostic rate was 5%, 28.33%, 95% and 13.33%. TB-IGRA showed the highest positive rate and sensitivity, and TB-DNA-PCR represented the highest omission diagnostic rate. The sensitivity of TB-IGRA + PPD was 98.33%, while the omission diagnostic rate was 51.67%. The sensitivity of TB-IGRA + TB-DNA-PCR was 95%, while the omission diagnostic rate was 5%. The sensitivity of TB-IGRA + ADA was 100%, while the omission diagnostic rate was 0%. In addition, the TB-IGRA + ADA had the highest sensitivity and the lowest omission diagnostic rate. Conclusion: TB-IGRA has high positive rate, high sensitivity and low omission diagnostic rate, which is superior to the traditional sputum test for tuberculosis. Notably, the combination of PPD, TB-DNA-PCR, ADA is capable of improving the diagnosis rate, and the diagnosis rate can reach 100% when combined with ADA, which is able to provide solid diagnostic value in clinical practice.

利用PCR技术检测肾结核患者血和尿中TB-DNA的结果及分析

笔者利用ＰＣＲ技术检测了５例疑似肾结核患者血和尿中的ＴＢ－ＤＮＡ。结果显示所有患者的尿中ＴＢ－ＤＮＡ均阳性，其中４例患者的血中ＴＢ－ＤＮＡ也阳性。血和尿中ＴＢ－ＤＮＡ均阳性可能会为临床上确诊为结核提供可靠依据。

荧光定量PCR对痰涂片阴性肺结核的临床诊断价值

目的探讨荧光定量PCR检测对痰涂片阴性的肺结核的诊断价值。方法采集180例痰涂片阴性的肺结核病患者及100例非结核性肺部疾病患者(肺炎、肺癌及矽肺患者)的支气管灌洗液(BALF),应用荧光定量PCR检测BALF中的TB-DNA,同时进行BALF结核杆菌培养及抗酸染色法检查结核杆菌,比较不同方法对肺结核的临床诊断效率。结果肺结核组中BALF中TB-DNA阳性检出率为62.77%,显著高于BALF抗酸染色法的阳性检出率33.89%(P<0.05),与BALF结核杆菌培养法无明显统计学差异(P>0.05),治疗1个月后TB-DNA的阳性检出率也高于抗酸染色法(P<0.05)。结论荧光PCR定量检测TB-DNA能提高结核杆菌的检出率,可作为诊断及判断疗效的方法。

利用PCR技术检测537份新疆结核病人痰标本中的结核分枝杆菌

目的 提高聚合酶链反应在检测人结核分枝杆菌中的特异性和敏感性。方法 在聚合酶链反应中对 4种DNA片段即结核杆菌特异插入序列IS6 110 ,IS10 81,和 16SrRNA ,6 5kDa摸板进行了比较 ;为获取较多的细菌DNA ,临床痰标本处理采用了国际标准化方法主要包括用一定量的N -乙酰 -L半胱胺酸和氢氧化钠处理痰标本 ;改进了RCR混和液的成份即添加了甘油 ,dUTP -尿嘧啶糖基化酶。结果 选择IS6 110作为结核杆菌特异性摸板用于检测细菌DNA ,制备出了 6批人结核杆菌PCR检测试剂盒 ,在对来自新疆结核病研究所的 5 37份痰标本的检测中发现 ,该试剂盒检测的特异性为 6 5 .97% ,敏感性为 93 .5 3 %。结论改良TB -PCR试剂盒显示有较高的敏感性 ,特异性还有待于进一步完善 ,该试剂盒在临床诊断中有一定的价值。

T-SPOT.TB在肺结核早期诊断中的应用研究

目的探讨结核感染T淋巴细胞酶联免疫斑点试验(tuberculosis T lymphocytes enzyme-linked immune SPOT test,T-SPOT.TB)在肺结核早期诊断中的应用价值。方法采用T-SPOT.TB,荧光定量PCR(fluorescence quantitative PCR,RQ-PCR)、结核蛋白芯片(TB-Ab protein chip)及结核菌素试验(purified protein derivative,PPD)检测189例疑似肺结核患者是否有结核菌感染。结果 T-SPOT,PCR,TB-AB和PPD方法的敏感度分别为91.54%(119/130),73.85%(96/130),63.08%(82/130)及57.69%(75/130),特异度分别为89.83%(53/59),86.44%(51/59),67.79%(40/59)及66.10%(39/59);T-SPOT.TB方法敏感度均高于荧光定量PCR法等三种方法(x^2=14.216~41.573,均P<0.05),TSPOT.TB方法特异度均高于PPD试验及结核蛋白芯片方法(x^2=8.577~9.669,P<0.05);T-SPOT.TB,荧光定量PCR,TB-Ab及PPD阳性预测值分别为95.2%(119/125),92.3%(96/104),81.2%(82/101)和78.9%(75/95).而阴性预测值分别为82.8%(53/64),60%(51/85),45.5%(40/88)和41.5%(39/94);四种检测方法的假阳性率(误诊率)分别为10.2%(6/59),13.6%(8/59),32.2%(19/59)和33.9%(20/59),而假阴性率(漏诊率)分别为8.5%(11/130),26.2%(34/130),36.9%(48/130)和42.3%(55/130);四种检测方法的阴性似然比分别为0.11,0.16,0.48和0.51,而阳性似然比分别为9.0,5.4,2.0和1.7;四种检测方法的诊断符合率分别为91.0%(172/189),77.8%(147/189),64.6%(122/189)和60.3%(114/189);T-SPOT.TB,荧光定量PCR,TB-Ab及PPD的约登指数分别为0.81,0.60,0.31和0.24。结论在实验室检测早期结核菌感染的方法中T-SPOT.TB检测法是较敏感且特异度较高的方法,对不同阶段肺结核诊断更具临床应用价值。

35例支气管肺泡灌洗液结核杆菌PCR分析

对35例未能排除肺结核病患者,经纤支镜检查后回收支气管肺泡灌洗液(BALF),井取样行结核杆菌PCR(简称 BALF TB—PCR)检查,结果发现,16例结核病中,其 BALF TB—PCR(+)者11例,阳性率69%;而非结核病的19例中,其 BALF TB—PCR(+)者4例,提示 BALF TB—PCR 检查可作为结核病的辅助诊断。

微滴数字PCR定量检测全血样品中结核分枝杆菌特异CFP10基因

目的 采用微滴数字PCR技术（droplet digital PCR,dd PCR）检测全血中结核分枝杆菌特异性CFP10（10-k Da culture filtrate protein,CFP10）基因的拷贝数含量。方法 收集10例活动性肺结核患者（active tuberculosis,a TB）、10例肺外结核患者（extrapulmonary tuberculosis,EPTB）和5例健康对照者（healthy donars,HDs）全血并提取DNA,优化引物最佳扩增条件并制定重组质粒标准曲线,对样本中CFP10基因的拷贝数含量分别用dd PCR与实时定量PCR（quantitative real-time PCR,q PCR）进行检测和比较。结果 对重组质粒的灵敏度检测显示dd PCR技术明显优于q PCR技术。对样本的检测显示q PCR技术检测a TB、EPTB患者与健康人的全血中CFP10含量之间无统计学意义;但是dd PCR技术检测a TB、EPTB患者与健康人之间有显著统计学意义（P〈0.0001）;dd PCR检测a TB和EPTB患者（共20例）CFP10基因绝对定量的浓度约为3.4~94.0 copies/μL。结论 本研究首次报道采用dd PCR技术能灵敏地用于TB患者全血的诊断。

不同临床标本实时荧光定量 PCR诊断结核分枝杆菌的检出率差异研究

目的 讨论不同临床标本中结核分枝杆菌的检出率有何差异.方法 对1 343例疑似结核病患者的呼吸道和体液标本进行实时荧光定量PCR检测,同时对其中393例进行血清结核抗体检测.结果 1 343例疑似结核病患者不同临床标本经实时荧光定量PCR检测显示,支气管灌洗液检出率最高为57.14%,其次为支气管刷检物21.21%,尿液13.33%,痰液11.23%,胸腔积液9.09%,最低为腹腔积液3.85%.痰液与支气管灌洗液、支气管刷检物之间检出率差异具有统计学意义（χ2=14.32,5.87,P〈0.05）;393例不同临床标本同时进行实时荧光定量PCR和血清结核抗体检测,实时荧光定量PCR检出率为24.43%,血清结核抗体检出率为47.58%,两种方法的阳性率差异有统计学意义（χ2=44.73,P〈0.01）.血清结核抗体与实时荧光定量PCR检测的符合率为61.57%.结论 结核分枝杆菌在支气管灌洗液检出率最高,其次为支气管刷检物、尿液、痰液、胸腔积液,最低为腹腔积液.

TB-SA基因检测在结核病诊断的临床应用研究

目的研究结核分枝杆菌特异性基因(Tuberculosis-Specific AntigenTB-SA)在结核病诊断中的应用价值。方法选择2004年4—9月期间在成都市结核病防治院的住院结核病患者371例。其中肺结核307例;肺外结核64例(结核性胸膜炎47例、结核性腹膜炎7例、结核性脑膜炎10例);非结核病的呼吸系统疾病患者61例(肺炎4例、肺癌9例,急性支气管炎7例、慢性支气管炎14例、哮喘10例,COPD12例、肺间质纤维化1例、支气管扩张3例、矽肺1例);同时选择无结核疾患健康志愿者40例,全部选例均知情同意。入选肺结核病例和健康选择痰液、肺外结核病例选择胸腔积液、腹腔积液、脑脊液等标本进行抗酸杆菌浓缩集菌,结核杆菌培养和TB-SA基因扩增(PCR)。结果307例肺结核患者中PCR检测TB-SA阳性259例,敏感性为84.4%(259/307);细菌学培养阳性193例,敏感性62.9%(193/307);痰浓缩集菌镜检阳性95例,敏感性30.9%(95/307)。64例肺外结核患者中,TB-SA阳性35例;敏感性54.7%(35/64);核杆菌培养阳性5例,敏感性7.8%(5/64)。61例非结核呼吸系统病患者中,TB-SA阳性2例,特异性96.7%(59/61)痰涂片无阳性病例。40例健康志愿者,TB-SA及痰涂片均无阳性出现,特异性达100%(40/40)。结论TB-SA基因检测的特异性及敏感性都取得了满意的效果。操作也较为简单。

实时荧光PCR与BACTEC MGIT 960快速培养在初治痰涂片阴性未受抗结核治疗的肺结核诊断中的应用

目的:探讨实时荧光PCR与BACTEC MGIT 960分枝杆菌快速培养(快速培养法)准确诊断初治痰涂片阴性(涂阴)且未经受抗结核治疗的肺结核可能性。方法:实验组:368份临床诊断活动性肺结核患者的初治涂片阴性痰标本;对照组:55份非结核性肺部疾病患者的痰液标本。用FQ-PCR技术、快速培养法及改良罗氏培养参考法对两组标本进行分析,观察指标为TB-DNA和结核菌阳性率。结果:实验组和对照标本中FQPCR技术、快速培养法和改良罗氏培养参考法检测的TB-DNA或结核菌阳性率分别为:16.85%、23.37%、22.01%和1%、0%、0%。三种方法分别平均耗时3h、9.6d和28d。以改良罗氏培养参考法为参照,FQ-PCR法和快速培养法的敏感性分别为69.1%和100%;特异性分别为91.2%和98.6%。结论:FQ-PCR和快速培养法均能提高初治涂阴肺结核患者的早期确诊率,但FQ-PCR的敏感性尚需进一步提高。

慢性肾衰患者并发结核病的诊断结核菌DNA-PCR与抗PPD-IgG的比较

慢性肾衰患者的结核病发生率高，肺外结核多见，缺乏特异的临床表现，故诊断较为困难。本研究检测了１１例慢性肾衰并发结核患者的血中结核菌ＤＮＡ－ＰＣＲ和血清抗ＰＰＤ～ＩｇＧ（ＥＬＩＳＡ），并检测了其中５例患者的渗出液（肺、腹水）及排泄物（尿，痰），结果表明，在血中，抗ＰＰＤ－ＩｇＧ的阳性率明显高于结核菌ＤＮＡ－ＰＣＲ，在渗出液或排泄物中，二者的阳性率大致相同．提示在尿毒症患者这一特定的人群中，血清或渗出液（排泄物）中抗ＰＰＤ－ＩｇＧ的检测对诊断结核有重要意义。

基于牛呼吸道合胞体病毒F基因TB GreenⅡ荧光定量PCR检测方法的建立

为了对牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)进行准确定量检测,针对BRSV F基因设计特异性引物,构建pMD19T-BRSV-gF重组质粒,以其为模板优化反应条件及反应体系,建立TB GreenⅡ荧光定量PCR方法并应用于临床BRSV检测。结果显示,针对BRSV F基因建立的TB GreenⅡ荧光定量PCR方法标准曲线线性关系良好,相关系数为0.9952;灵敏度高,BRSV最低检测限达3.9×10^(1) copies/μL;特异性良好,可特异性检测出BRSV,对牛传染性鼻气管炎病毒和牛病毒性腹泻病毒的扩增呈阴性;重复性好,批内和批间重复性试验变异系数均小于2%。对50份临床样品进行检测,荧光定量PCR对BRSV的阳性检出率(26%)明显优于常规PCR方法(14%)。结果表明针对BRSV F基因建立的TB GreenⅡ荧光定量PCR检测方法适合BRSV的临床检测。

Diagnosis of Bovine Tuberculosis by Comparative Intradermal Tuberculin Test, Interferon Gamma Assay and <i>esxB</i>(CFP-10) PCR in Blood and Lymph Node Aspirates

Bovine tuberculosis (TB) is a chronic debilitating disease of huge economic importance due to loss in production, morbidity and mortality, and has a potential zoonotic threat. TB is endemic in India and has a worldwide prevalence, therefore, needing early diagnostic technique for the eradication of TB globally. Currently, compared to the eradication programme of TB in Medical sector, Veterinary sector is lagging behind though TB is one of the major zoonotic diseases prevalent in dairy animals and wildlife in India. With the “End TB” strategy by WHO in human, parallel measures for early diagnosis and culling has to be followed in case of animals for an overall successful eradication programme. The objective of this study is diagnosis of TB in cattle and buffaloes by using the cell-mediated immune response tests, i.e. Comparative Intradermal Tuberculin Test (CITT) and Interferon gamma (IFN-γ) assay, and Polymerase Chain Reaction (PCR) targeting esxB gene (CFP-10 protein) and to compare their diagnostic capabilities. This study was carried out in 202 dairy cattle and buffaloes from an organized dairy farm, where almost all of the animals appeared clinically healthy. We found that, the combined use of both CITT and IFN-γ assay lead to more accurate diagnosis of TB, although IFN-γ assay was more specific than CITT. However, esxB PCR showed almost similar sensitivity to IFN-γ assay and may be used as a fast alternative method for the diagnosis of bovine TB from blood samples.