实时荧光定量PCR检测莫氏立克次体

目的 采用TaqMan-MGB探针建立检测莫氏立克次体的实时荧光定量PCR.方法 依据莫氏立克次体外膜蛋白B基因(ompB)设计引物和探针,以克隆的ompB作模板建立实时荧光定量PCR方法.结果 建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.995);该方法能检出＜10拷贝的莫氏立克次体DNA,敏感性为普通PCR的1 000倍.用该方法检测莫氏立克次体DNA,结果为阳性,但是检测其他相关细菌DNA的结果均为阴性.用该方法检测莫氏立克次体感染豚鼠血标本,50%样本检测为阳性,而普通PCR检测结果均为阴性.结论 该实时荧光定量PCR具有很高的特异性和敏感性,适合于快速检测样本中微量莫氏立克次体DNA,可用于临床实验室快速确诊地方性斑疹伤寒.     

实时荧光定量PCR法检测炭疽杆菌

为建立一种简便而特异的炭疽杆菌检测方法用作临床诊断工具，以nXO1持粒上的pag基因及pXO2质粒上的cap基因为靶合成特异引物，用DNA嵌入荧光染料SYBR Green I进行定量PCR扩增，在PCR后进行熔融曲线分析以确定得到的产物是否为目的产物，定量PCR条件优化结果为：引物浓度0．8umol/L,Mg^2 5.0mmol/L。该法检测炭疽的灵敏度达10^3拷贝，并能区分炭疽杆菌与其他蜡样杆菌，表明实时荧光定量PCR技术能够快速准确，特异地对炭疽进行定量分析。     

实时荧光定量PCR检测应力性骨折新兵外周血TNFRSF10C mRNA表达

目的:检测TNFRSF10C基因在应力性骨折(SF)患者与正常人之间的表达差异。方法:采用1:1配比的病例对照研究,选取入伍新兵SF病例及正常对照各10例,取受试者空腹肘静脉血,采用实时荧光定量PCR检测TNFRSF10C基因表达,结合管家基因GAPDH进行相对定量分析。结果:设计并合成了一组只特异性结合人TNFRSF10C基因的实时荧光定量PCR引物,结果未发现非特异性扩增。10例SF样本中有7例TNFRSF10C基因表达上调,上调1.4倍至6.7倍,另3例SF样本TNFRSF10C基因表达下调,分别为50%、90%和90%。结论:本研究建立的TNFRSF10C基因实时荧光定量PCR检测体系特异性好、可靠。TNFRSF10C基因表达结果为分析SF发病的分子机制及SF早期诊断提供了重要线索。     

霍乱弧菌O139实时荧光PCR检测方法的建立及应用

目的：建立检测霍乱弧菌O139的实时荧光PCR检测方法，为霍乱弧菌0139的检测提供快速、敏感、特异的检测手段。方法：用复合探针法荧光PCR检测技术，检测0139特异基因——编码糖基转移酶的基因。结果：本方法特异性好，检测非0139肠道菌均无响应；灵敏度高，可检测低至10CFU的细菌；重复性较好，批间批内变异系数均小于3％。对采自浙江省0139霍乱弧菌感染患者的335份标本进行检测，共有133份为阳性，而采用常规细菌培养法，仅检测出90份阳性。结论：本方法的建立为霍乱的诊断提供了又一有效手段。     

Hendra病毒的实时PCR检测方法的建立

目的：建立针对Hendra病毒的敏感、特异和快速的实时PCR法，为Hendra病的早期诊断提供依据。方法：采用常规PCR法和实时PCR法分别对构建的Hendra病毒全长G基因的重组质粒DNA进行扩增，并比较两种方法的敏感性。结果与结论：应用常规PCR方法可检测到100fg/μl的含Hendra病毒特异序列的重组质粒DNA，而实时PCR法则可检测到0．1fg/μl的DNA，后者的敏感性比常规PCR法的提高了1000倍，该方法不但操作上更加简便、快速，还可避免交叉污染，适于对Hendra病毒特异序列的检测。     

实时荧光定量PCR测定HBV-DNA的方法和临床意义

HBV—DNA定量是目前诊断乙型肝炎、判断病毒复制和评价患者肝脏病变、预后及药物疗效的重要指标之一，但在指导诊疗时仍需结合血清标记物、肝损害指标乃至HBV基因分型。HBV—DNA定量的方法有PCR核酸探针杂交法、R．ELISA、竞争定量PCR、实时荧光定量PCR，本文综述定量检测HBV—DNA意义及PCR检测方法。     

探讨实时荧光PCR法检测乙肝病毒DNA在临床中的应用

目的:探讨实时荧光PCR法检测乙肝病毒DNA在临床中的应用.方法:对168例临床乙肝病毒携带者的血清标本用ELISA法进行定性检测,再用实时荧光PCR定量检测,对两种检测结果进行相关性分析.血清标志物的检测顺序设定为:(1)HBsAg;(2)HbsAb ;(3)HbeAg;(4)HbeAb;(5)HbcAb;(6)PreS1 -Ag.结果:几种乙肝模式中,"大三阳"患者的DNA阳性检出率和拷贝数均比其他模式高,并有显著性差异.PreS1-Ag的阳性率与乙肝DNA的阳性率有很好的一致性,但由于两者检测的成分和表达的意义不完全一致,因此不能等同于或代替HBV-DNA的检测.结论:PCR定量测定HBV-DNA可以真实地反映体内乙肝病毒感染和复制及病毒载量情况,更有利于临床治疗和疗效的观察.并且实时荧光PCR法检测HBV-DNA具有快速、准确等优点,而且对低复制持续感染乙肝病人也能诊断.     

脂血和溶血对HBV DNA实时荧光定量PCR检测的影响

目的 探讨溶血和脂血两种因素对HBV DNA实时荧光定量PCR方法 的影响.方法 (1) 收集临床HBV DNA标本,按HBV DNA浓度分为Ⅰ水平、Ⅱ水平;收集HBV DNA阴性的重度乳糜状脂血标本和正常人标本.将以上标本按不同比例混合,制作成极高TG浓度不同HBV DNA水平:AⅠ、AⅡ组;高TG浓度不同HBV DNA水平:BⅠ、BⅡ组;正常TG浓度不同HBV DNA水平:CⅠ、CⅡ组.实时荧光定量PCR扩增.(2)收集临床HBV DNA标本,每人采集3份血标本,其中2份标本进行-20 ℃冷冻不同时间,制成重度溶血DⅠ、DⅡ组;中度溶血EⅠ、EⅡ组;无溶血对照组FⅠ、FⅡ组.实时荧光定量PCR扩增.(3)利用方差分析各检测结果 之间是否有统计学差异.结果 不同浓度脂血和溶血,对两种水平HBV DNA荧光定量PCR检测结果 均无统计学差异.结论 溶血和脂血对实时荧光定量PCR检测HBV DNA结果 无影响.     

代谢综合征相关新基因MSRG实时荧光PCR检测方法的建立

目的 建立测定MSRG mRNA表达量的实时荧光定量PCR检测方法,为新基因的功能研究奠定基础.方法 根据MSRG cDNA序列设计荧光PCR适用的引物和探针,构建质粒标准品,建立标准曲线用于荧光PCR相对定量,检测荧光PCR方法的灵敏性、特异性和重复性.荧光PCR检测不同浓度葡萄糖[5 6mmol/L(G5.6)、22mmol/L(G22)、33.3mmol/L(G33.3)]刺激人肝癌细胞株HepG2细胞MSRG的表达并与半定量RT-PCR方法进行比较.结果 建立了MSRG mRNA表达的实时荧光PCR检测方法.荧光PCR检测显示G33.3、G22和G5.6组HepG2细胞MSRG表达均有显著差异,半定量RT-PCR方法检测G33.3组MSRG表达显著增加,但不能检测出G22和G5.6组间有差异.结论 本实验建立的MSRG mRNA表达实时荧光PCR检测方法灵敏性、特异性和重复性较好,为MSRG功能的研究提供了一种重要手段.     

副溶血弧菌实时荧光定量PCR快速检测方法的建立

目的 建立一种快速检测副溶血弧菌的实时荧光定量PCR方法.方法 根据副溶血弧菌tdh基因设计合成引物及TaqMan探针,利用阳性质粒和模拟标本建立副溶血弧菌实时荧光定量PCR检测方法.结果 以副溶血弧菌DNA为模板克隆了tdh基因,得到阳性质粒.利用阳性质粒建立了定量PCR方法,得到标准曲线y=-4.9197x+58.72,决定系数(R2)为0.9864.此方法具有很好的特异性,在对15种肠道致病菌的检测中,只有副溶血弧菌基因组DNA的扩增结果为阳性.对粪便和食物模拟标本进行检测,敏感性均为102cfu/μl.此方法重复性较好,对4种浓度(105、104、103、102cfu/μl)的菌液各进行3次检测,结果均为阳性,且Ct值的变异系数＜2%.结论 建立了一种检测副溶血弧菌的荧光定量PCR方法,该法的敏感性和特异均较好,适用于快速、准确地检测食品和粪便中的沙门菌.     

Identification of Francisella tularensis using real-time fluorescence polymerase chain reaction

A Francisella tularensis-specific, TaqMan probe-based, real-time fluorescence polymerase chain reaction (PCR) assay required approximately 60 minutes and consistently achieved a sensitivity of < or = 10 fg of F. tularensis genomic DNA (five genome equivalents). Specificity testing against a genomic DNA cross-reaction panel comprised of 22 bacterial organisms representing closely related species, diverse genera, and human genomic DNA resulted in no false positives of significance. The assay was conducted on a field-deployable thermocycler, the R.A.P.I.D. ("Ruggedized" Advanced Pathogen Identification Device), a microbial identification system that can provide rapid and accurate identification F. tularensis.     

微孔杂交快速检测主要黄病毒方法的建立

目的建立特异、敏感、实用的登革病毒Ⅰ-Ⅳ型、流行性乙型脑炎病毒及黄热病毒检测及分型方法。方法在系统分析上述病毒基因组序列基础上,分别设计针对6种病毒的特异性包被探针,扩增、克隆、测序后包被聚乙烯微孔板。随后设计检测用PCR引物,并于引物5′端标记生物素,对待检样品进行扩增后用检测探针进行微孔杂交(PCR-ELISA)反应,并对包被DNA浓度、包被时间、杂交温度、杂交时间等反应条件进行优化。结果初步建立了微孔杂交快速检测上述病毒的方法,试验结果直观,阳性标本吸光度(A)值在0.5以上,阴性结果A值在0.1以下,S/N值在10.0以上。结论PCR-ELISA方法敏感、特异,为上述病毒的快速诊断提供了新的方法。     

Comparison of real-time polymerase chain reaction and conventional polymerase chain reaction methods for the rapid identification of Bacillus anthracis

Bacillus anthracis spores have been shown to be one of the most effective biological weapons. For the rapid detection of B. anthracis spores, several genetic markers, including chromosomal and plasmid-based sequences, were studied with polymerase chain reaction (PCR) methods. In the present study, a method using a primer/probe set based on the pXO1-encoded pag gene for the detection of B. anthracis was tested in addition to culture. Eight pathological samples (four blood-immersed cotton specimens, two spleen tissue specimens, and two blood smears) with confirmed positive results for anthrax were used. All samples were suspended in saline solution and fixed with Gram and Giemsa stains for examination of colony and capsule formation. Amplicons were analyzed on 2% agarose gels with the classic PCR method. For real-time PCR, a fluorescently labeled TaqMan probe was used with a Smartcycler. Positive smear and cotton samples were confirmed with the standard culture and real-time PCR methods, but the same samples were found to be negative with the classic PCR method. A spleen sample known to be positive for B. anthracis was found to be negative with the culture method because of possible contamination with Proteus-type bacteria.     

聚合酶链反应检测嗜肺军团菌的实验研究

 目的探讨并建立聚合酶链反应(PCR)用于检测嗜肺军团菌的方法.方法用PCR方法扩增嗜肺军团菌巨噬细胞感染增强子基因(mip)片段,琼脂糖凝胶电泳检测扩增产物;分析其特异性和敏感性.结果在嗜肺军团菌14个血清型参考株均扩增出特异的340bp片段,而三株非嗜肺军团菌及其他需鉴别诊断的常见病原菌均未扩增出此特异条带.检测灵敏度为2.6 pg/ul基因组DNA.结论依据mip基因建立的嗜肺军团菌PCR检测方法具有高度的敏感性和特异性,是诊断与鉴别诊断嗜肺军团菌感染的一种有效手段.     

A real-time fluorescence polymerase chain reaction assay for the identification of Yersinia pestis using a field-deployable thermocycler

Real-time fluorescence polymerase chain reaction is a microbial identification method that can provide rapid and accurate results using a field-deployable thermocycler, the RAPID ("ruggedized" advanced pathogen identification device). A Yersinia pestis-specific TaqMan assay required approximately 75 minutes and achieved a sensitivity of 100 fg of Y. pestis genomic DNA (20 genome equivalents). Specificity testing against a genomic DNA cross-reaction panel comprised of 22 bacterial species encountered in the respiratory tract resulted in no false positives. No cross-reaction occurred with human genomic DNA.     

Typing of Y chromosome single nucleotide polymorphisms in a Japanese population by a multiplexed single nucleotide primer extension reaction

We have developed a new method for typing single nucleotide polymorphisms (SNPs) on the human Y chromosome based on a multiplexed single nucleotide primer extension. This method has the advantage that several SNPs are typed rapidly and simultaneously. We examined 15 different SNP loci on Y chromosome, M9, M105, M122, M125, M128, M130, SRY465, IMS-JST006241, IMS-JST006841, IMS-JST002611, IMS-JST003305, IMS-JST008425, IMS-JST021354, IMS-JST021355 and IMS-JST055457, in 159 Japanese males. From the typing results of these 15 loci, we found 13 haplotypes. Gene diversity for each locus ranged from 0.025 to 0.486 and the haplotype diversity was estimated to be 0.838. This method could be readily applied for personal identification and paternity testing.     

Identification of Aedes aegypti and its respective life stages by real-time polymerase chain reaction

An Aedes aegypti-specific, fluorogenic probe hydrolysis (Taq-Man), polymerase chain reaction assay was developed for real-time screening using a field-deployable thermocycler. Laboratory-based testing of A. aegypti, A. aegypti (Trinidad strain), Culex pipiens, Culex quinquefasciatus, Anopheles stephensi, and Ochlerotatus taeniorhynchus individual adult mosquitoes and mixed pools (n = 10) demonstrated 100% concordance in both in vitro sensitivity (six of six samples) and specificity (10 of 10 samples). A single adult A. aegypti was identified in a pool of 100 non-A. aegypti mosquitoes. The limit of detection of A. aegypti egg pools was five individual eggs. Field testing was conducted in central Honduras. An A. aegypti and Culex spp. panel of individual and mixed pools (n = 30) of adult mosquitoes, pupae, and larvae demonstrated 100% concordance in sensitivity (22 of 22 samples) and 97% concordance in specificity (29 of 30 samples), with one false-positive result. Field testing of an A. aegypti and Culex spp. blind panel (n = 16) consisting of individual and mixed pools of adult mosquitoes, pupae, and larvae demonstrated 90% concordance in sensitivity (nine of 10 samples) and 88% concordance in specificity (14 of 16 samples).     

Detection of T to G mutation at position 59 in the Lewis gene by mismatch polymerase chain reaction

Recently, we discovered the missense mutations of T to G at position 59 and of G to A at position 508 in one of the Lewis-negative (le) genes (Koda et al. 1993). In the present study, we report a method to detect the mutation at position 59 using mismatch PCR amplification and endonucleaseMspI digestion. For this mutation, we found that 7 out of 12 Lewis-negative, and none of 15 Lewis-positive individuals were homozygous, while 4 out of 12 Lewis-negative, and 4 out of 15 Lewis-positive individuals were heterozygous. These results indicate that the mutation at position 59 is a common mutation in thele genes.     

3种重要虫媒病毒的RT-PCR检测

目的：建立快速、敏感、特异的虫媒病毒RT-PCR检测方法。方法：采用改进的核酸制备方法，分别从感染的乳鼠脑和细胞培养上清液中提取病毒RNA，再用RT-PCR和套式PCR方法进行检测。结果：通过对肌PCR反应条件的优化，用3种虫媒病毒的特异引物均可从不同稀释度的感染小鼠脑和细胞上清中提取的病毒RNA扩增出特异的目的基因片段。甲病毒属的东部马脑炎病毒和西部马脑炎病毒之间，及与黄病毒属的黄热病毒均无交叉反应，表明建立的RT-PCR检测方法特异性强。套式PCR与肝PCR比较可提高检测敏感性10～10000倍。结论：建立的肝PCR和套式：PCR法可用于3种虫媒病毒的快速检测。