SARS冠状病毒实时荧光RT-PCR定量检测

为建立一种快速、准确、特异的SARS病毒RNA定量检测方法,根据复合探针荧光定量分析原理,对SARS病毒核酸进行实时荧光定量RT-PCR检测.借助计算机辅助,对SARS病毒基因靶序列以及检测引物和探针进行了优化筛选;利用体外转录SARS病毒RNA靶序列,对RT-PCR反应的镁离子浓度参数进行了优化;比较Trizol法、磁珠法、Qiagen法、煮沸法等4种方法提取RNA的检测效果,建立了样本处理方法;通过对构建的体外转录靶序列模型的检测,对本方法的灵敏度、特异性、定量线性关系、精确度等进行了评价,并通过对42例临床标本的检测对本方法的检测效果进行了评估. 通过克隆SARS病毒核酸靶序列并进行体外转录,获得了长度约1.2 kb的体外转录RNA靶序列;经优化,荧光RT-PCR反应液中的Mg²⁺浓度以4.0 mmol/L为最好;RNA提取方法采用磁珠法效果较好;本方法的检测灵敏度最低可达5个拷贝的体外转录RNA分子,特异性100%,C_t值的CV值小于5%.对临床确诊的42份SARS病人血清和漱口水标本的检测结果表明,该方法的检出率为79%,与荧光抗体检测法的符合率为70%.上述结果表明,该方法建立的荧光定量RT-PCR技术能够快速准确、特异、敏感地对SARS病毒核酸进行定量分析,为临床SARS冠状病毒RNA的检测提供了新的,更为有效的检测方法.     

TaqMan-MGB荧光定量RT-PCR技术快速检测H5亚型禽流感病毒

建立以TaqMan-MGB荧光探针为特点的荧光定量RT-PCR方法,用于检测H5亚型禽流感病毒.针对H5亚型禽流感病毒血凝素(HA)基因保守区域设计特异性引物与TaqMan-MGB荧光探针,筛选并优化荧光定量RT-PCR反应体系与反应条件,用以提高方法的特异性、敏感性与准确性;并通过体外克隆技术建立病毒基因拷贝数进行定量分析.结果表明引物与探针的优化浓度分别640nmol/L和480nmol/L,体系具有良好的保守性和特异性,与其他呼吸道病毒均无交叉反应.方法检测灵敏度为100拷贝/反应,标准曲线线性范围为107～102拷贝/反应,从病毒核酸提取至检测完成仅需3h左右,操作简便,重现性好.本研究建立的TaqMan-MGB荧光定量PCR方法特异、敏感、快速,适合于临床实验室进行H5亚型禽流感病毒的快速定量检测.     

TaqMan—MGB荧光定量RT-PCR技术快速检测H5亚型禽流感病毒

建立以TaqMan-MGB荧光探针为特点的荧光定量RT-PCR方法,用于检测H5亚型禽流感病毒。针对H5亚型禽流感病毒血凝素(HA)基因保守区域设计特异性引物与TaqMan-MGB荧光探针,筛选并优化荧光定量RT-PCR反应体系与反应条件,用以提高方法的特异性、敏感性与准确性;并通过体外克隆技术建立病毒基因拷贝数进行定量分析。结果表明:引物与探针的优化浓度分别640nmol/L和480nmol/L,体系具有良好的保守性和特异性,与其他呼吸道病毒均无交叉反应。方法检测灵敏度为100拷贝/反应,标准曲线线性范围为107～102拷贝/反应,从病毒核酸提取至检测完成仅需3h左右,操作简便,重现性好。本研究建立的TaqMan-MGB荧光定量PCR方法特异、敏感、快速,适合于临床实验室进行H5亚型禽流感病毒的快速定量检测。     

PCR产物中无错误碱基拷贝最低比率的计算

分析了PCR过程中带有错误碱基拷贝的量变过程,得出不同循环(n)后不同类型拷贝数的计算通式并以逐次代入方式归纳出PCR产物中无错误碱基拷贝最低比率(R)和有效循环数(N),拷贝酶促合成链长(H)及错配率(f)的关系式R_n=(1-Hf/2)^N-1(1-Hf),对PCR技术制备表达用DNA片段有指导意义.     

O1群霍乱弧菌实时荧光定量PCR快速检测方法的建立

目的:建立针对O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,并进行模拟粪便标本的检测评价。方法:根据O1群霍乱弧菌O抗原编码基因rfb的特异性序列设计引物和TaqMan探针,建立检测O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,对所建立的方法分别进行实验室内的灵敏度及特异性评价;将O1群霍乱弧菌灭活菌株悬液倍比稀释后与健康成人新鲜粪便混匀,制备成模拟带菌者粪便标本,提取DNA,进行Taq-Man PCR检测,用以评价该方法。结果:建立了快速检测O1群霍乱弧菌的实时荧光定量TaqMan PCR方法,灵敏度为每反应体系104拷贝;该方法对其他14种肠道菌DNA没有扩增;该方法对模拟粪便标本的检测灵敏度为每反应体系102 CFU。结论:建立了一种快速、高效检测O1群霍乱弧菌的荧光定量PCR检测方法,该方法可用于O1群霍乱弧菌临床粪便标本的检测。     

人偏肺病毒TaqMan-MGB探针实时定量RT-PCR检测方法的建立及初步应用

目的:建立人偏肺病毒(hMPV)核酸特异的快速、敏感的TaqMan-MGB探针实时定量RT-PCR检测方法.方法:分别设计hMPV特异的引物与荧光标记探针,合成hMPV绝对定量RNA模板,建立实时荧光定量PCR方法,并与常规RT-PCR平行比较,对其灵敏性、特异性和可重复性,以及用于临床样本的适用性等进行评价.结果:本方法可对hMPV进行特异性诊断,检测灵敏度可达10拷贝/25 μL,检测线性范围至少可达10 1～10 6拷贝/反应,且实验重复性好,初步应用于北京地区采集的158份临床鼻咽拭子标本,定量RT-PCR检出31份标本阳性,明显高于常规RT-PCR方法(22/158).结论:建立了人偏肺病毒TaqMan-MGB探针定量RT-PCR检测方法,并初步证实可用于临床鼻咽拭子标本的检测,为开展hMPV的流行监测及临床早期诊断提供了技术手段.     

基于双重芯片式数字PCR快速鉴定KPC型碳青霉烯耐药肺炎克雷伯菌的方法

【背景】由于碳青霉烯类药物的泛用和滥用,致使肺炎克雷伯菌碳青霉烯耐药株与日俱增,产碳青霉烯酶是肺炎克雷伯菌对碳青霉烯类药物耐药的主要原因。目前对肺炎克雷伯菌碳青霉烯耐药株的检测方法存在费时费力、特异性差、灵敏度低等问题。【目的】建立一种能同时检测肺炎克雷伯菌和碳青霉烯酶基因bla_KPC的双重芯片式数字PCR方法。【方法】依据肺炎克雷伯菌的特有基因yhaI和碳青霉烯耐药基因bla_KPC保守序列设计特异性引物和探针,确定双重芯片式数字PCR同时对yhaI和bla_KPC两个基因核酸浓度绝对定量的检测范围、检出限和最佳实验体系,并进行方法特异性、灵敏度、重复性分析及临床菌株的检测。【结果】双重芯片式数字PCR检测灵敏度比双重实时荧光定量PCR提高了约1.5个数量级,在两基因同时检出的情况下,最低检出限分别为3.74 copies/μL (yhaI基因)和1.93 copies/μL (bla_KPC基因);优化后的双重芯片式数字PCR对参考菌株检测特异性的结果与双重实时荧光定量PCR结果一致;利用优化后的双重芯片式数字PCR方法共检测58株临床菌株,其中肺炎克雷伯菌43株,属肺炎克雷伯菌且含有bla_KPC基因的菌株13株,这与质谱及耐药谱检测结果一致。【结论】利用双重芯片式数字PCR技术建立了产KPC型碳青霉烯酶肺炎克雷伯菌的绝对定量检测方法。该方法特异性强、灵敏度高、准确度好,可用于检测具有碳青霉烯酶基因bla_KPC的肺炎克雷伯菌的核酸检测和定量分析,也为产其他类型碳青霉烯酶的病原菌检测提供了新的技术参考。     

人博卡病毒TaqMan探针实时定量PCR检测方法的建立及初步应用

目的:建立人博卡病毒(HBoV)核酸特异、快速、敏感的TaqMan探针实时定量PCR检测方法,并对临床样本进行检测。方法:比对编码HBoV非结构蛋白NP-1的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量PCR检测方法,并与传统PCR方法进行比较,然后分别对两者的灵敏性、特异性、稳定性及临床样本检验的适用性等进行评价。结果:所建立的实时定量PCR检测方法可用于HBoV的特异性检测;相对于传统PCR所达到的250拷贝/反应的检测灵敏度,实时定量PCR的检测灵敏度可高达10拷贝/反应,检测范围为109～101拷贝/反应,且具有良好的特异性和重复性;初步用于76份临床呼吸道标本检测,检出阳性5例,高于普通PCR方法(3/76)。结论:建立了HBoV TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,为开展HBoV流行病学监测及早期临床诊断提供了技术手段。     

呼吸道合胞病毒TaqMan探针实时定量RT-PCR检测方法的建立及应用

目的:建立呼吸道合胞病毒(RSV)核酸特异、快速、敏感的TaqMan探针实时荧光定量PCR检测方法,并对临床样本进行检测。方法:比对编码RSV非编码蛋白的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量RT-PCR检测方法,并与传统RT-PCR方法进行比较,分别对两者的灵敏性、特异性、重复性及临床样本检验的适用性进行评价。结果:所建立的实时荧光定量RT-PCR检测方法可用于RSV的特异性检测。相对于传统RT-PCR方法100拷贝/反应的检测灵敏度,实时荧光定量RT-PCR的检测灵敏度达到10拷贝/反应,检测范围为1010~101拷贝/反应,且具有良好的特异性和重复性。从169份临床呼吸道标本中检出RSV阳性40例,高于普通PCR方法(31/169)。结论:建立了RSV的TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,在临床上具有较好的应用前景。     

猪链球菌和猪多杀性巴氏杆菌多重实时荧光PCR方法的建立

【背景】猪链球菌(Streptococcus suis,SS)和猪多杀性巴氏杆菌(Pasteurella multocida,Pm)都是能引起宿主致病的人畜共患病原菌,常出现混合感染,临床诊断上易与猪瘟、猪丹毒等混淆。【目的】快速、有效鉴别猪链球菌病和猪多杀性巴氏杆菌病,建立一种能同时检测2种病原的多重实时荧光定量PCR检测方法。【方法】基于猪链球菌的gdh基因和猪多杀性巴氏杆菌的plpE基因,设计2对特异引物及TaqMan探针,以细菌16S rRNA基因设计通用引物及探针,通过对反应条件优化,建立了一种能同时检测猪链球菌和猪多杀性巴氏杆菌的多重实时荧光定量PCR检测方法。【结果】该方法能够特异性地检测猪链球菌和猪多杀性巴氏杆菌,与细菌分离后的测序结果验证完全一致。此方法对重组质粒标准品的最低检出浓度分别为4.53×10²copies/μL和3.97×10²copies/μL。重复性试验结果显示,该方法的组内和组间变异系数均小于3%。【结论】本实验所建立的方法准确、简便、可靠,能够用于2种病原菌的同时检测,为猪链球菌病和猪多杀性巴氏杆菌病的防治提供了有效的检测工具,具有重要的流行病学意义和临床应用价值。     

新型Taq Man-MGB探针在结核分枝杆菌实时PCR检测中的应用

为建立一种比现有方法敏感、准确性高、重复性好的结核分枝杆菌DNA定性定量检测方法 ,以TaqMan探针技术为基础 ,运用TaqMan MGB探针 ,实时检测临床标本中的结核分枝杆菌DNA .用来自临床标本的DNA及克隆于载体的IS6 1 1 0序列检测所建立方法的有效性 .结果显示 ,所建立方法的最低检测限度为 1个基因拷贝 反应 ,在每反应 1 0 0 ～ 1 0 8拷贝范围内 ,Ct 值同DNA量的对数呈线性关系 .同一模板不同时间或同一时间不同管内扩增 ,所得Ct 值恒定 .用该方法检测 37例结核分枝杆菌培养阳性的痰液标本 ,敏感度为 1 0 0 % ;用该方法检测 1 6例TB系列阴性参考品 ,特异性为1 0 0 % .结果表明 ,所建立的方法是用于结核分枝杆菌定性定量检测较理想的方法     

Detection by multiplex polymerase chain reaction and typing of Chlamydia trachomatis isolates

Abstract The multiplex polymerase chain reaction (PCR) was applied for the detection of the Chlamydia trachomatis chromosome and plasmid. The multiplex PCR demonstrated a sensitivity of 0.8 fg of chlamydial DNA, corresponding to the detection of about 5 copies of the plasmid. Analysis of 195 genital specimens collected randomly from a female population, showed that the multiplex PCR is more sensitive and rapid than culturing for detecting Chlamydia trachomatis . Moreover, sequencing of the II variable domain of the ompl gene, directly from DNA of the clinical specimens, appears to be a simple and rapid method for determining serovar isolates.     

Quantification of Pyrenophora teres in infected barley leaves using real-time PCR

Net blotch is a barley foliar disease caused by two forms of Pyrenophora teres: Pyrenophora teres f. teres (PTT) and Pyrenophora teres f. maculata (PTM). To monitor and quantify their occurrence during the growing season, diagnostic system based on real-time PCR was developed. TaqMan MGB (Minor Groove Binder) primers and probes were designed that showed high specificity for each of the two forms of P. teres. As a host plant internal standard, TaqMan MGB primers and probe based on RacB gene sequence were designed. The method was optimised on pure fungal DNA and on plasmid standard dilutions. Quantification was accomplished by comparing Ct values of unknown samples with those obtained from plasmid standard dilutions. The assay detects down to five gene copies per reaction. It is able to produce reliable quantitative data over a range of six orders of magnitude. The developed assay was used to differentiate and quantify both forms of P. teres in infected barley leaves. Correlation R(2)=0.52 was obtained between the Ct values and size of symptoms areas in early stage of infection. Application of the TaqMan MGB technology to leaf samples collected in 20 barley varieties in the region Kromeriz during the growing season of 2003 and 2004 revealed that P. teres f. teres predominated in these 2 years. The developed method is an important tool to quantify and monitor the dynamics of the two forms of P. teres during the growing season.     

Detection of plasmid DNA from all Chlamydia trachomatis serovars with a two-step polymerase chain reaction.

A polymerase chain reaction was used to amplify a 137-base-pair sequence of DNA from a Chlamydia trachomatis plasmid. Various parameters of the polymerase chain reaction were explored, and it was found that two short steps per reaction cycle were sufficient to achieve 10(12)-fold amplification in less than 1 h. By use of this procedure, 10(-18) g of a sequence of plasmid DNA, representing the amount of that sequence found in one C. trachomatis bacterium, was amplified to the point where it was clearly visible on an ethidium bromide-stained polyacrylamide gel under UV light. DNA from intact cells from each of the 15 serovars of C. trachomatis could also be amplified for visualization. With this procedure, the presence or absence of C. trachomatis DNA in a sample could be established in less than 1.5 h. The speed and extreme sensitivity of this detection procedure may make it a useful method for the detection of C. trachomatis, and similar techniques should be possible for any type of bacteria.     

Detection and frequency of Chlamydia trachomatis DNA in synovial samples from Tunisian patients with reactive arthritis and undifferentiated oligoarthritis

We aimed to determine the frequency of Chlamydia trachomatis DNA in the synovial compartment of 34 arthritic patients. Chlamydia trachomatis DNA was detected using a nested PCR targeting the cryptic plasmid, the 16S rRNA gene and the outer membrane protein 1 gene. The presence of serum immunoglobulin (Ig)G and IgA antibodies against C. trachomatis was studied by a microimmunofluorescence assay and by an enzyme-linked immunosorbent assay, respectively. Synovial samples from 20 of 34 (59%) patients [nine with reactive arthritis (ReA), seven with undifferentiated oligoarthritis (UOA), two with rheumatoid arthritis and two with osteoarthritis] were positive for at least one C. trachomatis DNA sequence by nested PCR. The high sensitivity results most likely from the combination of a standardized automated MagNA Pure extraction method, PCR targeting three different C. trachomatis genes and the screening for C. trachomatis in synovial tissue and fluid samples. There was no correlation between the presence of C. trachomatis DNA in the joint and a Chlamydia -specific serologic response. Our data support that PCR is the method of choice to establish the diagnosis of Chlamydia -induced arthritis in patients with ReA. We suggest that this diagnosis might also be considered in C. trachomatis -positive patients previously classified as UOA.     

3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3′-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T_m) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5′-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T_m (65°C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T_m and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.     

Comparison of DNA extraction from cervical cells collected in PreservCyt solution for the amplification of Chlamydia trachomatis

OBJECTIVE: The aim of this study was to compare and evaluate three methods of DNA extraction for the amplification of Chlamydia trachomatis in uterine cervical samples collected in PreservCyt solution. ThinPrep is the trade name for the slide preparation. METHODS: Thirty-eight samples collected in LCx buffer medium, which were identified as C. trachomatis infected by ligase chain reaction (LCR), were selected for this study. DNA from the PreservCyt samples was extracted by three methods: (i) QIAamp kit, (ii) boiling in Tris-EDTA buffer with Chelex purification, and (iii) Proteinase K digestion with Chelex purification. Sample DNA was tested for the presence of C. trachomatis by PCR using cryptic plasmid research (CTP) primers and major outer membrane protein research momp gene (MOMP) primers. Real-time (LightCycler) PCR for relative C. trachomatis quantification following DNA extraction was performed using primers (Hsp 60) for the 60 kDa heat-shock protein hsp60 gene. RESULTS: Amplification using CTP primers was the most successful with each of the extraction protocols. Boiling in buffer was the least successful extraction method. QIAamp was the best extraction method, yielding the most positives with both the CTP and MOMP primers. Proteinase K-Chelex extraction gave similar sensitivity to QIAamp extraction with CTP primers but lower for MOMP primers. CONCLUSIONS: The DNA extraction method must be carefully selected to ensure that larger PCR amplicons can be successfully produced by PCR and to ensure high sensitivity of detection of C. trachomatis. In this study it was found that the QIAamp extraction method followed by PCR with the CTP primers was the most successful for amplification of C. trachomatis DNA.     

血清E型沙眼衣原体的Real-time PCR定量分析

目的沙眼衣原体感染是最常见的性传播疾病,本文拟建立一种准确快速、标准化的感染动物组织衣原体载量检测体系。方法体外扩增感染用沙眼衣原体血清E型,克隆衣原体特异基因OMP1基因片段作为标准品,用Real time PCR法测定衣原体基因组拷贝数进行衣原体定量。结果 Real time PCR在OMP1基因片段200至2×108拷贝检测结果成线性,在模板中加入小鼠基因组未出现非特异扩增,同时未影响扩增效率。结论针对衣原体特异基因OMP1的实时定量PCR方法可以较为灵敏的特异的定量检测感染动物样本中的衣原体。     

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.