乙型肝炎病毒感染产妇乳汁HBV DNA定量检测的意义

目的探讨乙型肝炎病毒（HBV）感染产妇乳汁HBV DNA定量检测的意义。方法应用荧光定量聚合酶链反应（PCR）技术检测HBV感染产妇乳汁HBV DNA。结果142例HBV感染产妇乳汁HBVDNA定量阴性90例,HBVDNA小于1．00×10^3copy／mL,阳性52例,HBV DNA大于1．00×10^3copy／mL（36．6％）,其定量范围1．21×10^3～5．19×10^3copy／mL,平均为1．56×10^4copy／mL。结论HBV感染产妇乳汁HBV DNA定量检测意义不大,不能作为判断是否可以母乳喂养的依据。     

铜川地区3660例乙型肝炎患者HBV定性与定量结果分析

目的通过对乙型肝炎（下称乙肝）病毒（HBV）定性与定量的分析,了解两种结果的相互关系及HBV定量的临床诊断价值。方法对3660例乙肝患者血清标本用ELISA法进行定性检测,并依据乙肝两对半定性结果进行归类分组,再用实时荧光PCR定量检测。结果2151例HBsAg（＋）、HBeAg（＋）、抗-HBc（＋）组中有2018例HBVDNA定量拷贝数大于1.00×10^3copy/mL,阳性率为93.82%;1026例HBsAg（＋）、抗-HBe（＋）、抗-HBc（＋）组中有352例HBVDNA定量拷贝数大于1.00×10^3copy/mL,阳性率达到34.3%;483例HBsAg（＋）、抗-HBc（＋）组中有19例HBVDNA定量拷贝数大于1.00×10^3copy/mL,阳性率为3.9%。结论HBV感染者在定性检测＂两对半＂的同时,检测HBV定量对临床诊断治疗和疗效观测更有意义。     

乙型肝炎病毒e抗原检测评价慢性乙型肝炎患者体内病毒复制的探讨

目的探讨乙型肝炎病毒e抗原（HBeAg）检测在评价乙型肝炎（下称乙肝）病毒复制中的价值。方法对湛江市妇幼保健院肝病专科306例慢性乙肝患者用酶联免疫吸附试验（ELISA）法进行血清学标志物检测和实时定量聚合酶链反应检测HBV DNA。结果125例HBeAg阳性标本中HBV DNA含量超过10^3copy／mL的有108例，占86．4％；181例HBeAg阴性标本中HBV DNA含量低于10^3copy／mL有101例，占55．8％。结论HBeAg阳性一般可以认为病毒在体内复制，但HBeAg阴性不能认为病毒在体内处于静止状态。HBV DNA定量测定是评价乙肝体内复制最可靠的指标。     

乙型肝炎患者HBV DNA定量与血清标志物的关系分析

目的分析乙型肝炎病毒（HBV）血清标志物与HBV DNA定量之间的关系。方法分别采用酶联免疫吸附试验（ELISA）和荧光定量PCR（FQ-PCR）对391例乙型肝炎表面抗原（HBsAg）阳性血清进行检测,分析检出率及相关性。结果391例HBsAg阳性血清共检出7种模式,以HBV DNA含量大于5×10^2copy／mL为阳性。HBsAg阳性（＋）、乙型肝炎e抗原（HBeAg）（＋）、乙型肝炎核心抗体（抗-HBc）（＋）模式的阳性检出率较高,为96．9％,HBV DNA含量也较高,其中大于10^7copy／mL的血清为53．8％。HBsAg（＋）、抗-HBe（＋）、抗-HBc（＋）模式中阳性检出率为25．3％,171例（74．7％）HBV DNA含量小于5×10^2copy／mL。HBsAg（＋）、抗-HBc（＋）模式中阳性检出率为65．4％。结论HBsAg和HBV DNA联合检测,更能反映乙型肝炎患者HBV感染、传染性及体内复制情况。     

非洲赤道几内亚比奥科岛疟原虫感染的检测方法比较

目的:比较非洲赤道几内亚比奥科岛疟原虫检测方法的差异。方法:2010年6月至2012年7月,赤道几内亚比奥科岛马拉博医院就诊的220例疟疾患者纳入研究。应用荧光定量PCR、快速免疫法和镜检法进行检测。结果:PCR法检出率显著高于镜检法(P0.05)。三种检测方法在虫种鉴别方面无显著统计学差异(P0.05)。结论:荧光定量PCR法可用于疟原虫感染的检测。荧光定量PCR法检出率优于镜检法。     

乙型肝炎病毒实时荧光定量PCR检测方法的建立

目的建立一种采用Lightcycler系统进行HBVDNA实时荧光定量PCR的方法,并探讨其临床应用价值。方法针对HBV基因组S区设计一对扩增引物,并通过预实验严格优化反应体系的组成和条件;将T载体与HBVRT区扩增后纯化的产物进行连接反应,然后转染大肠杆菌（DH5a）,经蓝、白斑筛选后挑取阳性菌落,提取质粒,制备外标准品。结果用于制成外标准品的质粒经1：10的缓冲液倍比稀释,制作标准曲线,线性方程为：Y=-3．344X＋37（r2=0．9999）;通过检测已知浓度并经倍比稀释的HBVDNA,表明其最低检测限为5×10^2 IU／mL;拷贝数介于5×10^2～5×10^8 IU／mI。之间的HBVDNA浓度与ct值具有良好的线性关系。结论外标法实时荧光定量PCR是一种定量相对准确、灵敏度高、特异性强、操作相对简便的方法;该方法可用于乙型肝炎患者病情监测,有效指导临床用药;联合该法与血清标志物检测,可更准确评价HBV感染者病情。     

聚合酶链反应-测序技术快速检测耐异烟肼结核分枝杆菌KatG基因突变的研究

目的应用PCR-DNA测序技术快速检测耐异烟肼结核分枝杆菌分离株KatG基因突变,评价其在检测结核分枝杆菌异烟肼耐药性方面的应用价值。方法47株耐异烟肼结核分枝杆菌临床分离株及30株结核分枝杆菌敏感分离株用PCR-DNA测序技术检测KatG基因突变。结果47株耐异烟肼结核分枝杆菌分离株中,有31株KatG基因检出有突变,突变检出率为66.0%(31/47);30株结核分枝杆菌敏感株检出1株KatG基因突变。结论PCR-DNA测序技术方法敏感、准确、特异,可快速检测结核分枝杆菌KatG耐药基因突变,有利于耐异烟肼结核分枝杆菌耐药性的快速检测。     

聚合酶链反应-测序技术快速检测耐异烟肼结核分枝杆菌KatG基因突变的研究

目的 应用PCR-DNA测序技术快速检测耐异烟肼结核分枝杆菌分离株KatG基因突变,评价其在检测结核分枝杆菌异烟肼耐药性方面的应用价值.方法 47株耐异烟肼结核分枝杆菌临床分离株及30株结核分枝杆菌敏感分离株用PCR-DNA测序技术检测KatG基因突变.结果 47株耐异烟肼结核分枝杆菌分离株中,有31株KatG基因检出有突变,突变检出率为66.0%(31/47);30株结核分枝杆菌敏感株检出1株KatG基因突变.结论 PCR-DNA测序技术方法敏感、准确、特异,可快速检测结核分枝杆菌KatG耐药基因突变,有利于耐异烟肼结核分枝杆菌耐药性的快速检测.     

基因芯片技术在乙型肝炎病毒耐药和分型检测中的价值

目的通过基因芯片技术快速检测临床血清样本中乙型肝炎病毒的基因亚型和耐药突变情况。方法同时用荧光定量PCR和基因芯片2种方法检测211例临床慢性HBV感染血清样本,并用DNA测序对基因芯片HBV分型和耐药结果进行验证。结果211例样本荧光PCR定量结果均大于或等于5．0×10^2IU／mL,基因芯片检出阳性210例,未检出的1例样本定量值小于1．0×10^3Iu／mL。210例基因芯片检测阳性的样本,基因芯片检测显示耐药突变病例67例,占31．9％;基因亚型2种,其中B型137例,占65．2％,c型69例,占32．9％,B、C混合感染4例,占1．9％。上述210例样本经DNA测序验证,基因亚型和耐药突变类型完全符合。结论基因芯片技术具有准确、灵敏、高通量的特点,适用于临床乙型肝炎病毒基因分型和耐药突变检测。     

实时荧光定量PCR法用于人类SLC01B1和ApoE基因多态性检测的方法学评价

目的：探讨在ISO15189实验室认可体系下,实时荧光定量 PCR法检测人类SLC01B1和ApoE基因多态性的方法学评价。方法本文收集经Sanger测序确定基因型的20份DNA样本。采用实时荧光定量PCR法对其中2份样本的SLCO1B1*1b、SLCO1B1*5、ApoE2和ApoE4共4个多态性位点分别批内重复扩增检测10次,以评价方法的精密度;再使用该方法扩增所有样本的4个多态性位点,每个位点检测一次,并与Sanger测序法结果比对,以评价方法的准确性;使用实时荧光定量PCR法对其中1份经梯度稀释的DNA样本进行扩增,以评价方法的最低检测限。结果2份DNA样本扩增所得Ct值变异系数均小于2．5％（≤1％）;与Sanger测序法结果对比,实时荧光定PCR法结果准确性达100％（20／20）;DNA浓度在0．964 ng／μl时,该方法仍可正确检出其基因型。结论借助经典实时荧光定量PCR法,我们能准确、快速地检测临床样本SLC01B1和ApoE基因多态性,用于指导正确合理使用他汀类药物。     

SYBR GreenⅠ荧光PCR熔解曲线鉴定鸟分枝杆菌方法的建立及初步应用

目的基于SYBR GreenⅠ荧光染料建立鉴定鸟分枝杆菌(Mycobacterium avium,MA)的荧光PCR熔解曲线法,为临床鉴定MA提供简单、准确性高的分子生物学检测方法。方法以MA的特异性插入序列IS1311为检测靶标设计引物,在60℃的退火温度下,建立鉴定MA的SYBR GreenⅠ荧光PCR熔解曲线方法,并对MA等21种分枝杆菌标准株和金黄色葡萄球菌等5种常见致病菌进行检测,评价方法的特异性、检测限;以hsp65、16S rDNA普通PCR扩增产物测序分析为“金标准”,鉴定200株分枝杆菌临床分离株,评估建立方法鉴定MA的准确性。结果SYBR GreenⅠ荧光PCR熔解曲线方法在30个循环内能准确鉴定出21种分枝杆菌标准株和5种致病菌中的MA,检测限为5.6×10-2 ng/μL。200株分枝杆菌临床分离株中,以PCR测序鉴定出16株MA和184株其他分枝杆菌,以SYBR GreenⅠ荧光PCR熔解曲线方法鉴定出15株MA和185株非MA;本研究建立的方法和测序方法对MA的鉴定率差异无统计学意义(P>0.05);两法符合率为99.5%(199/200),一致性较高(Kappa值>0.75,P<0.01);建立方法的敏感性为93.8%(15/16),特异性为100.0%(184/184),阳性预测值为100.0%(15/15),阴性预测值为99.5%(184/185)。结论以MA特异性重复序列IS1311为基础建立的SYBR GreenⅠ荧光PCR熔解曲线方法,具有较高的灵敏度和特异性,为MA的临床检测提供新的途径。     

Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples

Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen.A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T_m = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C_q) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea.The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples.     

Multiplex real-time SYBR Green I PCR assays for simultaneous detection of 15 common enteric pathogens in stool samples

Diarrheal diseases account for more than 50% of foodborne diseases worldwide, the majority of which occur in infants and young children. The traditional bacterial detection method is complex and time-consuming; therefore, it is necessary to establish a rapid and convenient detection method that can detect multiple pathogens simultaneously. In this study, we developed a set of five multiplex real-time SYBR Green I PCR assays to simultaneously detect 15 common enteric pathogens based on the Homo-Tag Assisted Non-Dimer system. These assays effectively reduced primer-dimer formation and improved the stability, uniformity, and amplification efficiency of multiplex PCR. The detection limit of the multiplex SYBR Green I PCR system was approximately 10⁴–10⁶ CFU/mL for stool specimens. Furthermore, we vitrified heat-unstable components on the cap of a reaction tube, showing that Taq DNA polymerase, dNTPs, primers, and SYBR Green I remained stable at 25 °C. In summary, we developed multiplex SYBR Green I PCR assays that can simultaneously detect 15 enteric pathogens. This method is comprehensive, rapid, inexpensive, accurate, and simple and displays high specificity.     

两种定量聚合酶链反应模式检测乳腺癌组织中缓激肽释放酶表达的比较

目的比较两种荧光定量聚合酶链反应（FQ-PCR）检测乳腺癌组织中激肽释放酶6（KLK6）基因的表达。方法分别用TaqMan探针和SYBR Green I荧光染料作为荧光指示剂,实时荧光定量PCR检测KLK6在乳腺癌中的相对表达水平,应用统计软件分析两种检测模式的差异。结果两种检测模式的检测结果均显示乳腺癌中KLK6的表达均高于正常组织,KLK6的相对表达水平无显著性差异。结论在扩增产物特异的条件下应用SYBR Green Ⅰ检测的效果同Taq Man探针,SYBR Green Ⅰ染料应用范围广泛,成拳低。     

建立RT-ARMS-qPCR系统定量测定含A1555G突变的线粒体DNA拷贝数

目的建立RT-ARMS-qPCR（real time-amplification refractory mutation system-quantitative PCR）系统定量测定含线粒体DNA（mitochondrial DNA,mtDNA）A1555G位点突变的片段的拷贝数,为阐明线粒体耳聋临床表型多样性的分子生物学基础奠定基础。方法根据ARMS的基本原理设计引物,在引物3’端插入错配碱基AC建立RT-ARMS-qPCR系统,优化定量PCR体系,并进行方法学评价。经PCR分别扩增相应突变型和野生型的片段,将其克隆到pGEM-T Easy载体上,构建质粒标准品;同时对含突变型和/或野生型mtDNA 1555位点的片段的拷贝数进行定量检测。结果RT-ARMS-qPCR系统用于检测含mtDNA 1555位点的片段,线性检测范围为102-108拷贝数/μl,检测灵敏度为1×102拷贝数/μl,其批内变异系数（CV）为1.34%,批间CV为1.96%,重复性好;突变型和野生型引物分别只扩增相应片段,特异性好。结论RT-ARMS-qPCR系统适合于定量检测含mtDNA A1555G点突变的线粒体DNA片段,结果稳定、准确,有助于阐明线粒体耳聋严重程度的分子基础。     

RT-ARMS-qPCR定量测定线粒体耳聋患者mtDNA A1555G位点突变

目的 探讨RT-ARMS-qPCR(real time amplification refractory mutation system quantitativePCR)系统定量测定线粒体DNA(mitochondrial DNA,mtDNA)A1555G位点突变在线粒体耳聋发病机制研究中的价值.方法 以PeR扩增含mtDNA 1555位点的片段,并将其克隆到pGEM-T Easy载体上,构建质粒标准品;在引物3'端插入错配碱基AC建立RT-ARMS-qPCR系统,对含突变型和野生型mtDNA 1555位点片段的12例线粒体耳聋患者进行定量检测.结果 RT-ARMS-qPCR系统在检测1个含野生型mtDNA 1555的重组质粒DNA模板时,其批内变异系数(CV)为1.34%,批间CV为1.96%,线性范围为102-108拷贝/ul;突变型或野生型引物只特异扩增相对应的序列,特异性好;突变型/野生型mtDNA拷贝数及突变型所占的百分比与耳聋的严重程度相关(r=0.771,P=0.003),突变型mtDNA所占的比例越高,耳聋程度越严重.结论 RT-ARMS-qPCR系统适合于定量检测mtDNAA1555G点突变的线粒体DNA片段,结果特异、稳定、准确.线粒体耳聋的严重程度与含突变型和野生型mtDNA 1555位点的拷贝数比例有关.     

不同分期胃癌患者外周血淋巴细胞Foxp3 mRNA表达水平的研究

目的 采用SYBR GreenⅠ实时荧光定量PCR,研究不同分期胃癌患者外周血淋巴细胞Foxp3 mRNA的表达水平.方法 普通RT-PCR结合胶回收制备Foxp3 mRNA-CDS标准品,利用标准品制备定量PCR标准曲线,对曲线进行灵敏度、精密度和特异性分析,用SYBR GreenⅠ实时荧光定量PCR检测40例胃癌和8例正常对照外周血Foxp3 mRNA.结果 获得Foxp3全长CDS标准品,标准曲线范围（2.8×10^2）～（2.8×10^8）拷贝/μl,最低检测值280拷贝/μl,批内和批间变异系数（CV）＜5%.Foxp3基因在胃癌患者外周血表达水平显著高于健康对照（P＜0.01）,表达水平与肿瘤分期显著相关（P＜0.05）.结论 Foxp3基因上调表达与胃癌的发生、发展和预后有关.     

SYBR Green Ⅰ实时荧光PCR检测survivin甲基化状态

目的建立SYBR Green Ⅰ实时荧光聚合酶链反应（PCR）检测survivin甲基化的方法。方法25例胃癌组织标本及与其配对的正常胃组织经甲基化敏感性限制性内切酶HpaⅡ和MspⅠ处理后，再用SYBR GreenⅠ实时荧光PCR对survivin外显子1进行检测。实时荧光定量PCR检测survivin基因内含子2，用于监测经限制性酶消化的基因组DNA浓度变化，10倍系列稀释的基因组标准物用于检验实时PCR的敏感度，PCR产物通过凝胶电泳分析证实。结果经Hpa Ⅱ酶切后，甲基化的目的基因PCR产物在熔解曲线上有Tm值为（91．5&#177;0．5）℃的峰，电泳证实为338bp的条带。所有经酶消化的样本都能扩增出以Tm值（79．5&#177;0．5）℃为特征的对照基因（survivin基因内含子2），表明基因组DNA未产生严重的非特异性降解。SYBR GreenⅠ实时荧光PCR检测甲基化目的基因的灵敏度为10^0拷贝／μL。用以上新建的体系检测25例胃癌标本，发现其survivin基因外显子1的去甲基化频率为96％。结论SYBR GreenⅠ荧光PCR法具有快速、准确、敏感、实时、简单和敏感的特点，是检测survivin外显子1甲基化状态的一种可靠的新方法。     

Rapid detection of qnr and qepA plasmid-mediated quinolone resistance genes using real-time PCR

Plasmid-mediated quinolone resistance genes in clinical strains cannot be detected by phenotypic traits but require gene detection. We developed a multiplex real-time polymerase chain reaction (PCR) assay using high-resolution melting master mix with ResoLight dye to detect qnr genes and a simplex real-time PCR assay using SYBR Green I to detect qepA genes. Using qnr-positive and qepA1-positive control strains, the ResoLight method was able to rapidly identify qnrA, qnrB, qnrS, qnrC, and qnrD genes; the SYBR Green I method identified qepA genes. Among 118 extended spectrum beta-lactamase-producing Enterobacteriaceae isolates, the 2 new assays efficiently detected and identified qnr in 9 strains, but no qepA gene. To our knowledge, this is the first study describing the detection of all 5 qnr and qepA genes using real-time PCR. The 2 tests constitute a significant step forward for screening for plasmid quinolone resistance genes in clinical strains.     

Quantitative and qualitative analyses of the SNRPN gene using real-time PCR with melting curve analysis

Prader-Willi syndrome and Angelman syndrome are distinct neurodevelopmental disorders that are associated with the deletion of the chromosomal 15q11-13 region or uniparental disomy of chromosome 15. In this article, we applied SYBR Green I-based real-time PCR and melting curve analysis assay for rapid genotyping of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene methylation status and for detecting aberrations in copy number in a single tube. A single pair of primers was designed to create a 357 bp fragment containing the cytosine phosphodiester guanine islands in the SNRPN promoter and to amplify both unmethylated and methylated sequences. Genotypes were identified based on the TC value for copy number changes and the characteristic melting temperature of methylated cytosine phosphodiester guanine. Genotyping of SNRPN was performed on blood samples of 20 individuals with Prader-Willi syndrome, 3 individuals with Angelman syndrome, and 20 unaffected individuals. The promoter methylation status and the copy number changes were successfully determined and compared with standard methylation-specific PCR, and were validated by multiplex ligation-dependent probe amplification. This single-tube, SYBR Green I, real-time PCR with melting curve assay is rapid, reliable, sensitive, and easy to perform. It is suitable for high-throughput analysis as an alternative technique for quantitative and qualitative analysis of target genes.