实时荧光定量PCR检测幼儿腹泻腺病毒

目的:建立实时荧光定量PCR方法,并检测腺病毒Ad40或Ad41感染的粪便标本。方法:用T-A克隆技术构建含腺病毒基因的载体作为标准模板,采用Taqman探针标记技术,建立实时荧光定量PCR方法。采集幼儿腹泻标本248例,分别用直接免疫荧光法和实时荧光定量PCR检测腺病毒Ad40和Ad41,比较两种方法的阳性检出率。结果:实时荧光定量PCR灵敏度和准确度(95.60%和96.80%)均高于直接免疫荧光法(78.5%和82.40%)(P0.05),而两者特异度差异无统计学意义(97.30%vs 96.70%,P0.05)。248例待检样本,直接免疫荧光法检出率为2.42%(6/248),实时荧光定量PCR检出率为7.66%(19/248),明显高于直接免疫荧光法(χ2=3.675,P0.05)。结论:成功建立实时荧光定量PCR检测腺病毒Ad40或Ad41方法。其灵敏度、准确度和阳性检出率经均优于直接免疫荧光法,且简便快速。     

人线粒体DNA荧光定量PCR检测方法的建立

建立SYBR Green I实时荧光PCR定量检测人线粒体DNA的方法。选取人线粒体DNA高度保守基因片段,将该基因片段与pCF-T载体连接后,转化入E.coli DH5α,提取重组质粒PCR及测序鉴定后,作为阳性模板建立SYBR-Green I荧光定量PCR标准曲线和熔解曲线。结果表明:构建的标准曲线线性关系良好(反应体系中含101~108拷贝时,扩增反应CT值与拷贝数的对数成线性关系),相关系数为0.997。批内和批间重复性测定的变异系数分别为1.23%~3.29%以及3.10%~5.21%。我们成功建立了实时荧光定量PCR检测人线粒体DNA的方法,该方法可作为进一步研究线粒体DNA的方法,在相关疾病诊断和监测中具有一定的应用前景。     

利用荧光定量PCR技术对人肠道乳酸杆菌进行定量检测

目的应用荧光定量PCR技术对人粪便内乳酸杆菌进行定量检测,建立乳酸杆菌的荧光定量PCR检测体系。方法依据人肠道乳酸杆菌16S rDNA序列设计属特异性引物,应用荧光定量PCR技术检测乳酸杆菌的16S rDNA,对粪便中的乳酸杆菌进行定量检测和分析,并与用传统方法所获得的结果进行比较。结果荧光定量PCR检测乳酸杆菌和传统方法检测乳酸杆菌获得的结果接近,差异无统计学意义（P〉0.05）。结论荧光定量PCR技术比传统方法省时、省力,且敏感性和特异性更高。     

登革热病毒RNA实时荧光定量RT-PCR检测方法的建立

目的建立TaqMan探针实时荧光定量RT-PCR方法,测定登革热病毒（DV）及DV病毒的RNA拷贝数。方法利用TaqMan探针,建立实时荧光定量RT-PCR方法,通过对登革热病毒RNA定量外标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量RT-PCR方法的灵敏度、特异性和重复性。结果该方法检测灵敏度可达1×103copies/mL,特异性及重复性良好,对同一样品进行5次重复检测,其循环阈值的平均标准偏差为0.792。结论TaqMan探针实时荧光定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定登革热病毒及DVRNA载量。     

人类疱疹病毒6型荧光定量PCR检测方法的建立和应用

目的 建立人类疱疹病毒6型(HHV-6)荧光定量PCR检测方法,并检测临床样本.方法 根据文献合成HHV-6 U65-66基因片段的特异性引物和TaqMan探针,构建质粒制备标准品,评估该方法的特异性、灵敏度和重复性;并用该方法检测93份临床诊断为病毒性脑炎的脑脊液标本.结果 本实验检测HHV-6的灵敏度为3×10(0)拷贝/μl;标准曲线间线性关系(R2)为0.999,扩增效率为97.9％;同一样本重复检测3次,组内Ct值的变异系数最大为0.61％,组间为3.13％;特异性检测中只有HHV-6阳性标本出现扩增曲线.93份临床标本中检出HHV-6阳性2例,检出率为2.15％.结论 本实验所建立的荧光定量PCR检测HHV-6的方法特异强、灵敏高、重复性好,具有应用于临床检测的潜在价值.     

实时荧光PCR方法快速检测空肠弯曲菌方法的建立

目的建立实时荧光PCR快速检测空肠弯曲菌的方法。方法以空肠弯曲杆菌HipO基因的保守序列为模板设计特异性引物探针,建立一种能快速检测样本中空肠弯曲杆菌的实时荧光PCR方法;对方法的特异性和敏感性进行评价,并以正常人粪便为空白样本,添加一定量空肠弯曲菌标准株菌液进行检测,以对方法的检测效果进行初步评价。结果该实时荧光PCR方法只对空肠弯曲杆菌进行特异扩增,同种属的结肠弯曲菌及其他常见食源性病原菌均不能扩增;整个检测过程只需要80min,对空肠弯曲菌菌悬液可检测至5个细菌,对加标粪便样本可检测至10-100个细菌。结论本研究建立的实时荧光PCR检测空肠弯曲菌方法不仅能实现对空弯菌的快速检测,而且还为空弯菌的快速诊断及其引起的食源性疾病的监控溯源提供有意义的参考。     

一种快速检测人腺病毒的免疫层析分析方法的建立

目的:建立一种基于免疫层析法(Immunochromatographic assay, ICA)的快速简便的人腺病毒(Human adenoviruses,HAdVs)检测方法。方法:利用抗原结合和病毒斑点实验检测发现单克隆抗体3C11和7E6能特异性结合所有测试的人腺病毒,以这两个抗体建立ICA方法。使用培养病毒及1...     

肠道病毒71型实时荧光RT-PCR检测方法的建立及临床评价

目的利用实时荧光RT-PCR技术,并通过系统的分析性能和临床性能评价,建立一种早期快速检测肠道病毒71型（EV71）的方法。方法根据EV71基因组中编码衣壳蛋白VP1基因保守区序列设计一对引物和一条荧光探针,利用实时荧光RT-PCR技术建立了检测EV71的方法,并对扩增产物进行分析,同时进行灵敏度、特异性、精密度评价,在此基础上利用不同标本类型共1104例临床样本对本方法和RT-PCR方法进行对比分析。结果本方法可以特异性的检测EV71,而对种属相近的或引起症状相似的其他病毒均无交叉反应。本方法检测灵敏度达到9.22×102PFU/ml,不同浓度样本的Ct值的变异系数在1.4%～2.9%之间。1104例临床样本的研究显示本方法与RT-PCR方法检测结果的总符合率达到96.74%,阳性样本的检出率要高于RT-PCR方法。结论本方法检测EV71具有灵敏度高、特异性强、精密度高、快速简便的特点,并与RT-PCR方法具有很好的符合率,在手足口病的早期快速诊断和疫情监测方面具有很好的应用前景。     

建立人微小病毒B19的PCR检测方法

人微小病毒Ｂ１９是微小病毒科中唯一能感染人的病毒。能够提供Ｂ１９抗原的病毒体外培养系统尚未建成，因此限制了血清学实验的开发和普及。为此作者建立起Ｂ１９病毒的ＰＣＲ检测方法。设计引物在表达外壳蛋白ＶＰ１基因区，扩增长度为４００ｂｐ。     

耐甲氧西林葡萄球菌荧光定量PCR检测方法的实验研究

目的建立一种简便、特异的mecA基因荧光定量PCR检测方法，用于耐甲氧西林葡萄球菌（MRS）的快速鉴定。方法以煮沸法快速制备DNA模板，采用SYBRGreenI随机参入法，建立mecA基因的实时荧光定量PCR检测体系。并对检测体系的敏感性、特异性和灵敏度进行评价。结果本法对纯菌落的检测敏感性和特异性分别为98．5％和96．9％，检测灵敏度可达10^1CFU／ml，最小检菌量约为3个菌／反应体系。结论本实验所设计的荧光定量PCR方法用于MRS的检测具有快捷、高敏感性、高特异性和高灵敏度的特点。适用于MRS的快速检测。     

Real Time PCR on Disposable PDMS Chip with a Miniaturized Thermal Cycler

This paper presents the design and implementation of a low-cost miniature PCR device consisting of a disposable reactor chip and a miniature thermal cycler. The simple fabrication of the PCR chip by PDMS (Polydimethylsiloxane) does not need micro-machining or photolithography processes. The thermal cycler was built with a thin film heater for heating and a fan for rapid cooling. This device can perform PCR tests in a single well chip or a multiple-well chip. It can run PCR reactions of different volumes to meet specific application requirements. The smallest reaction volume tested in this work is 0.9 μL. In addition, this device fits any standard fluorescence microscope for real time detection, which makes real time PCR affordable for most research labs and clinics with a fluorescence microscope. Real-time PCR of E. coli stx1 has been demonstrated with the device described.     

四种腺病毒核酸提取方法的比较

目的通过蛋白酶K法、苯酚法、病毒DNA／RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种腺病毒核酸提取方法的比较。为进一步研究腺病毒的分子生物学特性选择合适的方法提供参考。方法以腺病毒感染的A549细胞为样品．分别以蛋白酶K法、苯酚法、病毒DNA／RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种方法提取核酸．核酸经紫外分光光度计检测A260／A280的比值后,用荧光定量PCR方法检测核酸中腺病毒拷贝数浓度。并记录四种腺病毒核酸提取方法所需的操作时间。结果蛋白酶K法、苯酚法、病毒DNA／RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种方法提取核酸的A260／A280的比值依次为1．85532、1．7377、1．81474和1．43934,核酸中腺病毒拷贝数浓度依次为4．9×10^5copies／mL、3．94×10^3copies／mL、2．66×10^6copies／mL和6．15×10^6copies／mL,提取核酸所需的时间分别为1．5、15、0．5和0．5h。结论四种腺病毒核酸提取方法中,蛋白酶K法简单快捷、成本低廉,适合一般实验室使用;苯酚法适合用于提取细胞培养上清中病毒的核酸;病毒DNA／RNA试剂盒的优点是操作时间短,得到的病毒核酸纯度较高;腺病毒荧光PCR检测试剂盒对病毒的核酸损耗少．操作步骤少．适用于临床标本的腺病毒核酸检测。     

基于小波变换的QRS波群实时检测算法

本文研究了基于小波变换方法的心电信号QRS波群检测算法,通过对心电信号进行低通滤波、小波变换、差分平滑、阈值检测和修正策略等技术,提高了QRS波群的检测率.经MIT-BIH心律失常心电数据库全部48例数据的检验,QRS波检测灵敏度达99.82%,真阳性率达99.52%.在Windows环境下可实时实现.     

实时荧光定量PCR快速分子诊断唐氏综合征

目的　探讨快速诊断唐氏综合征的检测方法 .方法　采用实时荧光定量PCR技术 ,检测唐氏患者 2 3例、正常人 33例 ,定量分析比较ΔCt值 .结果　正常组和唐氏组ΔCt值有显著性差异 (p <0 .0 0 1) ,并建立了相应的参考值范围 .结论　实时荧光定量PCR具有简单、快速、特异性高等优点 ,可用于快速诊断唐氏综合征     

Detection of human orthopoxvirus infections and differentiation of smallpox virus with real‐time PCR

We developed a real‐time PCR protocol to detect orthopoxviruses (OPVs) from different clinical specimens and to separate variola virus from other OPVs. In our protocol, we used automated nucleic acid extraction system together with real‐time PCR to create a simple, safe and fast procedure to obtain an initial result. The sensitivity was better by using designed hybridization probes as compared to SYBR green I for detection. The detection limit ranged from 13 to 1,300 copies per 20 µl reaction volume depending on the sample type. The PCR detected all OPVs pathogenic to human (variola, cowpox, monkeypox, vaccinia) as well as camelpox and ectromelia viruses. Amplification of variola virus sequences could be distinguished from other OPVs by melting curve analysis. We also demonstrated the applicability of the assay in human cases of cowpox and vaccinia virus infections. J. Med. Virol. 81:146–152, 2009. © 2008 Wiley‐Liss, Inc.     

Detection of human cytomegalovirus DNA in perilymph of patients with sensorineural hearing loss using real-time PCR

Although cytomegalovirus (CMV) has been detected in the inner ear fluid of patients who succumbed to the complications of symptomatic congenital CMV infection, it has not been detected in the inner ear fluid of living patients. In this study, real-time polymerase chain reaction (PCR) was used to measure CMV DNA in clinical samples (including perilymph) collected from five patients with deafness. In case 1, diagnosed as a symptomatic congenital CMV infection, 3 copies/microl of CMV DNA were detected in perilymph, although no viral DNA was detected in peripheral blood mononuclear cells (PBMCs) or urine samples. In case 4, a suspected asymptomatic congenital CMV infection, 36 copies/microg of CMV DNA were detected in PBMCs, but neither perilymph nor urine contained viral DNA. Likewise, in case 5, a case of deafness of unknown origin, 48 copies/microg of CMV DNA were detected in the PBMCs, but none in the perilymph or urine. CMV DNA was not detected in the samples obtained from the remaining two cases with deafness of unknown etiology. To our knowledge, this is the first report to detect CMV DNA in an inner ear sample obtained from a living human subject.     

Quantification of adenovirus species B and C viremia by real-time PCR in adults and children undergoing stem cell transplantation

Adenovirus infection during stem cell transplantation is associated with high morbidity and mortality. Adenovirus species B and C have been the main causes for these infections; however, epidemiological details about the species are still unclear. To clarify the contributions of species B and C adenovirus, the DNA was tested serially by quantitative real-time PCR in peripheral blood, stool and urine of 32 patients (16 adults and 16 children) undergoing stem cell transplantation. Adenovirus species B viremia was detected in 10 of 16 adult and 6 of 16 pediatric transplant recipients. Adenovirus species C viremia was also detected simultaneously in five adult and three pediatric recipients. The stool and urine of patients with adenovirus viremia were also positive for the same adenovirus species as in blood. In contrast, in none of 50 healthy adult controls was adenovirus species B or C viremia detected. Among patients who developed adenovirus viremia, one adult recipient developed disseminated disease and died from multiple organ failure. The remaining patients experienced fever of several degrees and/or diarrhea during the period of adenovirus viremia; however, they all recovered without antiviral therapy. The results indicated that stem cell transplantation was frequently associated with adenovirus species B or C viremia, although it did not always cause serious infectious complications.