性病门诊中单纯疱疹病毒的荧光定量PCR检测与分析

单纯疱疹病毒(herpes simplex virus, HSV)系双链DNA病毒, 人类是其唯一的自然宿主.HSV主要引起生殖器疱疹(genital herpes, GH), 即感染泌尿生殖器及肛门部位的皮肤黏膜, 导致生产一种慢性、复发性的性传播疾病[1].GH的发生和复发可给患者带来很大的身心痛苦, 影响其生活质量.绝大多数GH由HSV-2引起, 目前尚无彻底治愈的方法[2],因此, GH已成为一个为人关注的危害人类健康的公共卫生问题.为了解本地区GH中HSV-2的感染现状, 我们对性传播疾病(STD)门诊中疑似GH的患者进行了HSV的定量检测, 现将结果报道如下.     

荧光定量PCR检测生殖器单纯疱疹病毒Ⅱ型DNA

目的为了调查单纯疱疹病毒Ⅱ型在男性和女性患者中的感染情况.方法用荧光定量PCR技术对59例男性和40例女性生殖器溃疡和疱疹患者进行HSVⅡDNA检测.结果男性平均年龄35.07±10.77,女性29.35±5.91,t=2.55,p=0.014;男性HSVⅡDNA阳性率为27.12%,定量拷贝对数为7.725±1.474,女性HSVⅡDNA阳性率为17.5%,定量拷贝时数为6.927±1.842(χ2=1.237,P=0.266;t=1.101,P=0.280).结论男性年龄高于女性年龄,男性和女性间感染阳性率无差异,急性感染的发生率也基本一致.     

荧光定量PCR检测4种常见性传播疾病DNA

目的调查我市近5年来4种常见性传播疾病(STD)的感染情况及流行情况。方法用荧光定量PCR技术对15481例可疑性传播疾病患者,对四种最主要的性传播疾病病原体———淋球菌、沙[衣原体、解脲支原体和人乳头状病毒进行检测,并进行详细的统计分析。结果近5年来4种常见性传播疾病病原体:沙[衣原体、解脲支原体和人乳头状病毒阳性率持续上升,淋球菌略有下降。淋球菌、沙[衣原体、解脲支原体和人乳头状病毒阳性率分别为15.90%、22.0%、36.57%、36.06%。结论性传播疾病已构成流行,其原因可能与混合感染和治疗不彻底有关,应引起全社会的高度重视。     

荧光定量PCR技术研究进展

基于荧光能量传递技术的荧光定量PCR技术可实时监测PCR反应 ,准确敏感地测定模板浓度及检测基因变异等 ,它的出现 ,极大地克服了原有PCR技术存在的不足如交叉污染等问题 ,并改善了PCR技术的应用如可广泛应用于临床观测患者病情发展及预后 ,判断药物疗效等。本文概述了目前几种荧光定量PCR技术即Taqman、Amplisensor、分子信标、Lightcycler及复合探针法的原理及特点。便于更好地理解及应用这项技术。     

荧光定量PCR技术及其应用

荧光定量PCR是新研制出的一种核酸定量技术.该技术在PCR反应系统中引入了荧光标记探针,具有高灵敏性、高特异性和高精确性的特点.目前已被应用于病原体测定、肿瘤基因检测、免疫分析、基因表达、突变和多态性研究等多个领域.     

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。     

荧光定量 PCR与半定量PCR检测HBV DNA的对比分析

目的：对照分析荧光定量PCR与半定量PCR检测血清HBV DNA的载量，为临床基因检测实验室提供方法学选择依据。 方法： 取164份临床检测标本各用荧光定量PCR与半定量PCR检测，结果作统计学分析。 结果: 两种检测方法的灵敏度和特异度没有显著差异（χ2 =1.75，P>0.05）。 结论: 临床基因检测实验室可用半定量PCR法替代荧光定量 PCR法检测血清HBV DNA的载量。     

荧光定量PCR法检测母乳巨细胞病毒感染和母婴传播

目的了解母乳人巨细胞病毒（HCMV）感染状况和母婴传播情况.方法应用荧光定量PCR（FQ-PCR）法检测390份母乳中HCMV-DNA含量,其中236份母乳配对与患儿血液或尿液中HCMV-DNA含量作一定分析.结果可疑或确诊HCMV感染患儿母亲母乳HCMV-DNA阳性率达71.28%,HCMV-DNA阳性母乳喂养的婴儿,其血或尿HCMV-DNA阳性率明显高于HCMV-DNA阴性母乳喂养的婴儿.结论HCMV感染母乳是婴儿获得性感染的主要途径.     

荧光定量PCR检测结果的报告方式探讨

目的 探讨荧光定量PCR检测结果的报告方式.方法 以荧光定量PCR检测乙型肝炎病毒DNA结果为例,用Poisson分布的概率函数公式计算一次抽样检测出现的三种结果的概率.结果 1次抽样Ct（阈循环数） ≥30时,总体起始拷贝数的95%可信区间上限是3.7;1次抽样Ct≤28,总体起始拷贝数为1的概率小于0.05（95%可信限）;1次抽样检测结果出现0时总体为1～8的概率之和是0.5818;2～6次抽样检测结果都出现0时,总体为1～8的概率之和分别是0.1565,0.0524,0.0187,0.0068,0.0025,要使报告血清中不存在HBV-DNA的可信度达95%以上（P＜0.05）,至少要进行4次抽样检测且Ct都≥30.结论 1次抽样检测Ct≥30时报告＂HBV-DNA≤4＊f拷贝/ml（Ct≥30）＂,其中f为抽样＂拷贝数/μl＂换算成报告拷贝数＂拷贝数/ml＂的换算因子;1次抽样检测Ct≤28时报告仪器自动计算的N值＊f;1次抽样检测28＜Ct＜30时,报告＂HBV-DNA＜4＊f拷贝/ml（Ct＝XX.X）建议追踪检查＂,其中XX.X为实测Ct值;报告单中参考范围应是＂HBV-DNA≤4＊f拷贝/ml（Ct≥30）＂.     

Monitoring of herpes simplex virus DNA types 1 and 2 viral load in cerebrospinal fluid by real‐time PCR in patients with herpes simplex encephalitis

A quantitative polymerase chain reaction (PCR) assay was evaluated retrospectively on 92 cerebrospinal fluid (CSF) samples from 29 patients with herpes simplex virus (HSV) encephalitis with the aim to study if the concentration of HSV genomes can be used as a prognostic marker and for monitoring of antiviral therapy. The results were compared to those obtained previously by nested PCR, and the numbers of HSV genomes/ml were evaluated in correlation to patient outcome and treatment. The aims were to compare the sensitivity of a conventional nested PCR to a quantitative PCR, to investigate the range of HSV genome concentration in initial samples and to evaluate possible relationships between the HSV DNA concentrations in CSF, neopterin levels, and outcome of disease. The 29 initial samples contained between 2 × 10² and 42 × 10⁶ HSV genomes/ml. There was no apparent correlation between the amount of HSV DNA in the initial samples and income status, initial neopterin levels, or prognosis. The number of HSV genomes/ml declined after treatment in all patients, but HSV DNA was still detectable after day 20 in 3 out of 16 patients. A long duration of genome detectability was found to correlate with poor outcome. There was no difference in sensitivity between the nested PCR and the quantitative PCR. While the quantitative PCR is more rational than a nested PCR, the quantitation of HSV genomes does not seem very useful as a prognostic marker in HSV encephalitis. J. Med. Virol. 81:1432–1437, 2009. © 2009 Wiley‐Liss, Inc.     

荧光定量聚合酶链反应检测乙型肝炎病毒DNA

目的 建立检测HBV病毒DNA的荧光定量PCR法（FQ-PCR）,并与运用常规凝胶电泳技术观察特异扩增带检测的结果加以比较。方法 合成扩增HBV DNA314bp特异保守序列的1对引物及1条带2个荧光基团的寡核苷酸探针。用PE-5700型定量PCR仪完成PCR反应及产物的荧光定量检测。同是PCR产物经琼脂糖凝胶电泳,EB染色,UVP（凝胶成像仪）检出有314bp带者为阳性或弱阳性。结果 建立了检测HBVDNA的荧光定量PCR技术,用已知HBV阳性模板不同拷贝数的标准溶液测得标准曲线Ct,原始拷贝数在10^5/ml以上者为阳性,用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性,阳性率29.2%;用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性,阳性率29.2%;用定性PCR观察到193例有阳性特异带,阳性检出率为27.65%。没有发现用定性PCR检测为阳性而用FQ-PCR检测为阴性者。结论 FQ-PCR检测HBVDNA较普通定性PCR技术具有操作简便,灵敏度更高、减少发生污染可导致假阳性结果的可能性及自动化程度高等优点,值得在临床检验中推广应用。     

Multiplex PCR for identification of herpes virus infections in adolescents

The aim of the study was to develop a multiplex PCR (mPCR) for a rapid and simultaneous detection of herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), and human cytomegalovirus (HCMV) DNA in squamous oral cells obtained from adolescents. Accuracy of the method was tested in a group of 513 adolescents, almost 11% of subjects were positive for infection with herpes viruses. Correlations with gender, age, and place of residence were sought. A similar incidence of HSV-2 and HCMV was found (4.3% and 5.4%, respectively) and the incidence of HSV-1 was the lowest (1%) in the study group. Conversely to HSV-2, HCMV was detected mostly in the youngest individuals. The same occurrence of all viruses was observed in boys and girls. The mPCR method described is suggested as a useful tool for epidemiologic studies of active herpes infections.     

肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究

目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100％.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测.     

Enhancing sensitivity of human herpes virus diagnosis with DNA microarrays using dendrimers

DNA microarray technology has become a promising new tool for the detection and identification of viral pathogens in human plasma and cell cultures. For exploration of this technology, we have developed DNA microarrays that encode capture oligonucleotide probes for different human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The on-chip hybridization is accomplished with the PCR amplicons of the respective human herpes virus types. In this original article, we attached multiple Cy3-fluorophores to the branched 5' ends of the labeling oligonucleotide primers. For the first time, we experimentally demonstrated that the self-designed, knowledge-based, and focused microarrays specifically hybridized to fluorophore-labeled pathogenic DNAs using dendrimer technology. The fluorescence signal enhancement via the dendrimers was up to 30 times compared with the quenched single Cy3-fluorophore-labeled HSV-1 DNA. The on-chip signal-amplifying effect depended upon the number of branches and the concentration of fluorophore-labeled pathogenic DNAs. Treblers were superior to doublers, as trebler-labeled nucleic acids had fluorescence-signal-enhancing effects over a broad range of labeled DNA concentrations exemplified for the quenched single Cy3-fluorophore-labeled HSV-1 and non-quenched single Cy3-fluorophore-labeled CMV DNAs.     

荧光定量PCR在筛查甲型流感病毒中的诊断价值

目的:探讨荧光定量PCR技术在筛查甲型流感病毒中的诊断价值。方法:用荧光定量RT-PCR方法检测150例疑似流感患者咽拭子中的甲型流感病毒RNA,用XT-1800全自动血液分析仪检测150例疑似流感患者EDTA抗凝全血中的白细胞计数,用胶体金法检测54例甲型流感病毒RNA阳性患者的甲型流感病毒核心蛋白。结果:在150例患者中,甲型流感病毒RNA检测阳性者有54例,阳性率为36%。54例甲型流感病毒RNA检测阳性者采用胶体金法检测甲型流感病毒核心蛋白,结果均为阴性。甲型流感病毒RNA检测阳性者白细胞计数为(6.81±2.12)×109/L,阴性者为(6.64±3.13)×109/L,白细胞计数在甲型流感患者和非甲型流感患者中无统计学差异(P0.05)。结论:荧光定量RT-PCR在检测甲型流感病毒RNA中有较好的阳性检出率,其敏感性和特异性明显优于胶体金方法和白细胞计数,能快速有效筛选出甲型流感患者,防止疫情爆发流行。     

Rapid detection of chromosome aneuploidies by quantitative fluorescence PCR: first application on 247 chorionic villus samples

We report the results of the first major study of applying quantitative fluorescence polymerase chain reaction (QF-PCR) assays for the detection of major chromosome numerical disorders. The QF-PCR tests were performed on a total of 247 chorionic villus samples, which were analysed blind, without any knowledge of the results obtained using conventional cytogenetic analysis. The aims of this investigation were to evaluate the detection power and accuracy of this approach by testing a large number of fetal samples and to assess the diagnostic value of each of the chromosome specific small tandem repeat (STR) markers used. In addition, we introduced three more markers specific for chromosomes 13, 18, and X to allow an accurate analysis of samples homozygous for a particular STR. Fluorescent labelled primers were used to amplify 12 STRs specific for chromosomes 21, 18, 13, X, and the amylogenin-like DNA sequence AMXY, expressed on the X and Y chromosomes. In this blind study of 247 fetal samples, 222 were correctly diagnosed by QF-PCR as normal for each of the five chromosomes investigated; 20 were diagnosed by QF-PCR as trisomic for chromosomes 21, 18, or 13, in agreement with the cytogenetic tests. Only one false negative result was observed, probably owing to the mishandling of the sample, which had been transferred through three laboratories before being analysed by QF-PCR. The 247 samples also included four cases of mosaicism or translocation; one case of mosaic trisomy 21 was detected by QF-PCR and the other cases were not identified by QF-PCR. The results of this investigation provide clear evidence that the QF-PCR assays are powerful adjuncts to conventional cytogenetic techniques and can be applied for the rapid and accurate prenatal diagnosis of the most frequent aneuploidies.     

Taqman探针实时荧光定量PCR检测肝脏疾病患者血清中miR-122的表达水平及其临床意义

 目的:运用Taqman探针实时荧光定量聚合酶链式反应（PCR）检测肝脏疾病患者血清中miR-122的表达水平并探讨其临床意义。方法:设计miR-122及U6 snRNA的茎环引物和Taqman探针,运用Taqman探针实时荧光定量PCR检测27例肝癌术前(HCC)患者、15例乙型肝炎（hepatitis B）患者、15例丙型肝炎（hepatitis C）患者、15例正常对照者（HC）、11例肝癌术后（PHCC）患者及10例肝癌术后复发（recurrence）患者血清中miR-122的表达水平,并分析miR-122与肝脏疾病相关标志物的关系。结果:Taqman探针实时荧光定量PCR方法能检测血清中miR-122的表达。HCC、hepatitis B、hepatitis C及recurrence患者血清中miR-122的表达水平均高于HC和PHCC患者（P<0.05）, hepatitis C患者血清miR-122表达水平高于HCC、hepatitis B和recurrence患者（P<0.05）,但HCC、hepatitis B和recurrence患者血清miR-122的表达水平无明显差异（P>0.05）,PHCC患者血清miR-122的表达水平比HCC和recurrence患者低（P<0.05）。血清乙型肝炎病毒表面抗原（HBsAg）阳性和(或)乙型肝炎病毒e抗原（HBeAg）阳性患者血清miR-122的表达水平高于阴性者（P<0.05）。血清丙型肝炎病毒抗体（HCV-Ab）阳性患者血清miR-122的表达水平高于阴性者（P<0.05）。血清丙氨酸氨基转移酶（ALT）与miR-122的表达水平有正相关性（r=0.34,P<0.05）。血清甲胎蛋白（AFP）≥400 μg/L组血清miR-122的表达高于AFP＜400 μg/L组（P<0.05）。结论: Taqman探针实时荧光定量PCR适用于检测血清miR-122的表达水平。在HCC、hepatitis B、hepatitis C及recurrence患者血清中miR-22均有不同程度的增高,尤其是hepatitis C患者,且PHCC患者血清miR-122表达下降,复发后升高。血清miR-122的表达与肝脏疾病的某些指标有关,提示血清miR-122可作为肝脏疾病,特别是肝癌早期诊断、手术疗效及预后判断的新指标。     

应用实时荧光定量PCR快速分子诊断唐氏综合征

目的探讨一种快速、准确诊断唐氏综合征的方法。方法采用实时荧光定量PCR技术，对25例唐氏患者、50名正常人外周血标本，扩增21号及1号、19号染色体上的多态位点，定量分析比较正常组及唐氏患者组的4对△Ct值。结果唐氏患者组△Ct值明显低于正常组，两组比较差异有统计学意义（P〈0．001）。初步建立了临床应用的参考值范围，可以有效区分出唐氏样本和正常样本。结论应用实时荧光定量PCR技术可快速、准确诊断唐氏综合征，为唐氏综合征的快速产前诊断开辟了新的途径。