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。     

运用基因芯片技术研究孕妇TORCH感染

本文利用DNA引物和探针设计软件DNAC lub和Prim er 5.0,根据从Genbank中查得的巨细胞病毒(HCMV169)、单纯疱疹病毒(HSV-Ⅰ,Ⅱ)、风疹病毒(RV)和弓形虫(TOX)基因序列分别设计16只寡核苷酸探针,长度30bp左右,合成后用简易手动点样仪M icroCaster按4×4矩阵点在氨基化玻片上,制成基因芯片;将被检测致病微生物DNA和质粒载体DNA用同一种内切酶切割、连接成“杂种”DNA,再利用载体上的通用引物进行PCR扩增和荧光染料标记扩增产物,并将该产物与DNA芯片杂交,用芯片扫描仪GeneTACTMUC4(Genom ic Solutions Inc)进行扫描,利用随机提供的软件GeneTAC IntegratorM icroarray Analysis Software Version3.30进行数据处理和分析。结果用该种方法制备的基因芯片可同时检测5种致病微生物,将用荧光定量PCR检测的阳性标本用基因芯片检测结果基本一致。     

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.     

肾移植后BK病毒感染者实时荧光定量PCR检测

背景：对于BK病毒感染、BK病毒相关性肾病的认识缺乏规范的实验室诊断程序和标准化的无创性检验方法。 目的：建立肾移植后患者尿液和外周血BK病毒感染负荷实时荧光定量PCR检测方法。 方法：针对BK病毒基因组,自主设计特异性引物BKV-F和BKV-R,Taqman荧光探针BKV-P,Taqman荧光探针BKV-P的5’端标记有荧光基团,在除5’端外的任意一个位置上标记有淬灭基团;然后处理待测样本,进行PCR反应。 结果与结论：将检测阳性的扩增产物进行基因测序,测序结果经BLAST比对后证实为BK病毒基因序列;利用上述方法对56份样本进行检测,其中,20份BK病毒血清样本及20份BK病毒尿液样本检测均为阳性,有S型扩增曲线。动态范围测定显示在103~1010 copies/mL之间标准曲线具有良好的相关性。5份健康人尿液样本,5份血液样本及6份临床常见的其他病原体血液样本检测均为阴性,无S型扩增曲线。结果表明该方法可进行定性、定量检测,特异性好,反应快速,一般30 min即可得到反应结果,并且成本低、假阳性少。     

实时荧光定量PCR在周围髓鞘蛋白22基因重复或缺失检测中的应用

目的 应用实时荧光定量PCR在腓骨肌萎缩症和遗传性压力易感性神经病患者中检测周围髓鞘蛋白22基因（peripheral myelin protein 22，PMP22）重复或缺失。方法 采用实时荧光定量PCR检测113个腓骨肌萎缩症家系先证者、4个遗传性压力易感性神经病家系先证者和50名正常人PMP22基因重复或缺失突变。结果 113个腓骨肌萎缩症家系中发现有36个存在PMP22基因重复，4个遗传性压力易感性神经病先证者均存在PMP22基因缺失，50名正常人未发现异常。结论 我国PMP22基因重复突变的致病频率为31．9％（36／113），PMP22基因缺失突变是遗传性压力易感性神经病常见的致病原因。     

荧光探针结合区序列突变对HBV DNA检测的影响

目的探讨标本荧光探针结合区序列突变对荧光定量PCR检测HBV DNA的影响。方法采用3种国产荧光定量PCR试剂对200例标本进行平行检测HBV DNA,分析3种试剂检测结果的差异,并对6例3种试剂检测结果无差异的标本和6例达安试剂检测结果明显降低或漏检标本进行测序。结果 3种试剂检测结果相差30倍以上的标本47例,且3种试剂均有不同程度的漏检;6例3种试剂检测结果无差异的标本荧光探针结合区序列无突变,6例达安试剂检测结果明显降低或漏检的标本荧光探针结合区序列均有突变。结论标本荧光探针结合区序列突变是造成达安试剂检测HBV DNA结果明显降低或漏检的原因。     

应用荧光定量PCR检测缺失型DMD携带者的研究

目的建立荧光定量PCR技术检测缺失型DMD携带者.方法应用9对引物扩增抗肌萎缩蛋白缺失热点区域,检测出常见的缺失型DMD患儿17例.用SYBR Green Ⅰ荧光定量PCR技术对缺失型DMD患儿家系中21例女性亲属进行检测.结果1例肯定携带者、11例可疑携带者和3例可能携带者证实为携带者;5例可疑携带者和1例可能携带者被排除.结论SYBR Green Ⅰ荧光定量PCR技术是一种检测缺失型DMD携带者的准确、快速、简便的方法.     

应用Affymetrix全基因组芯片检测染色体7q36区域的DNA拷贝数突变

目的 应用Affymetrix全基因组芯片结合荧光定量PCR(quantitative real-time PCR,qPCR)技术,进行致病性DNA拷贝数变异的精细定位研究.方法 以一个定位于染色体7q36的中国人遗传性三节拇指多并指综合征伴随Ⅳ型并指家系中的一例患者为研究对象.收集外周血标本,常规提取基因组DNA.应用Affymetrix Genome-Wide Human SNP Array 6.0芯片,将基因组DNA纯化,经过酶切、连接、扩增、标记、杂交、染色和扫描等步骤后得到原始数据,应用Affymetrix Genotyping Console 3.0软件进行拷贝数分析.在经芯片分析所确定的重复范围内设计引物,采用qPCR方法进行验证,并进一步缩小断端范围、精确重复区域范围.结果 将患者重复区域两断端范围由原来的113 kb和33 kb分别缩小到5.4 kb和1.8 kb,致病性DNA重复范围由原来的291～437 kb精确至379～387 kb.结论 应用Affymetrix全基因组芯片联合qPCR技术可以实现对DNA拷贝数突变的精确、可靠的检测.     

实时定量PCR在强直性肌营养不良1型重复突变检测中的应用

目的 鉴定一个强直性肌营养不良(dystrophy myotonica,DM)家系的致病突变,探讨实时定量PCR能否用于检测导致DM1的CTG重复延展突变.方法 采集家系成员外周血,提取基因组DNA.针对DM1位点DMPK基因内的CTG重复区和DM2位点CNBP基因内的CCTG重复区进行普通PCR、实时荧光定量PCR、微卫星标记连锁分析.结果 CTG重复区普通PCR产物电泳显示患者特有大于2 kb的弥散带.定量PCR显示,患者CTG重复区相对拷贝数约为0.5,CCTG重复区相对拷贝数约为1.在DM1位点的微卫星标记,患者均有共享等位基因;而在DM2位点的大部分标记,患者没有共享等位基因.结论 此DM家系的致病突变是DM1位点的CTG重复延展突变;应用实时定量PCR可以确定高度重复延展片段扩增失败,从而推断重复延展突变的存在,达到快速检测DM1的目的.     

实时定量PCR在强直性肌营养不良1型重复突变检测中的应用

目的 鉴定一个强直性肌营养不良(dystrophy myotonica,DM)家系的致病突变,探讨实时定量PCR能否用于检测导致DM1的CTG重复延展突变.方法 采集家系成员外周血,提取基因组DNA.针对DM1位点DMPK基因内的CTG重复区和DM2位点CNBP基因内的CCTG重复区进行普通PCR、实时荧光定量PCR、微卫星标记连锁分析.结果 CTG重复区普通PCR产物电泳显示患者特有大于2 kb的弥散带.定量PCR显示,患者CTG重复区相对拷贝数约为0.5,CCTG重复区相对拷贝数约为1.在DM1位点的微卫星标记,患者均有共享等位基因;而在DM2位点的大部分标记,患者没有共享等位基因.结论 此DM家系的致病突变是DM1位点的CTG重复延展突变;应用实时定量PCR可以确定高度重复延展片段扩增失败,从而推断重复延展突变的存在,达到快速检测DM1的目的.     

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。     

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。     

Quantitation of HCV RNA using real-time PCR and fluorimetry

Real-time PCR technology may provide an accurate and sensitive method to quantify hepatitis C virus (HCV) RNA. So far, studies have been carried out using the Taqman technology with the ABI Prism 7700 sequence detector. An alternative and simple real-time PCR assay is described with no probe requirement, based on the SYBR Green I dye and LightCycler fluorimeter. Amplicon synthesis was monitored continuously by SYBR Green I dye binding to double stranded DNA during PCR of the 5' HCV non-coding (NC) region. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with serial 10 fold dilutions of a modified synthetic HCV 5' NC RNA. A wide range linear relationship (up to 3.7x10(9) copies/ml) was observed between number of PCR cycle needed to detect a fluorescent signal and number of RNA copy. Intra- and inter-assay coefficients of variation were 0.7 to 2.1 and 3.7% respectively, indicating good reproducibility of the method. Thirty-three HCV positive sera of different genotypes were quantified by this method and gave similar but more sensitive results compared to the branched DNA (bDNA) technology.     

SYBR Green Ⅰ联合TaqMan荧光定量PCR检测HBV-DNA的意义

目的探索SYBR GreenⅠ联合TaqMan荧光定量PCR检测HBV-DNA的意义。方法选择浓度为10^8.48、10^5.70和10^3.70copies/ml的3种HBV-DNA阳性血清和〈1×10^3.0copies/ml的阴性血清各1份,在TaqMan-PCR混合反应体系中加入SYBR Green Ⅰ组成双荧光PCR（TaqMan＋SYBR Green Ⅰ组）,同时进行TaqMan和SYBR Green Ⅰ的单荧光PCR（分别为TaqMan组和SYBR Green Ⅰ组）,设置同一PCR和熔解曲线的循环参数,检测HBV-DNA含量及其Tm,每种方法一次检测每份血清5次。结果TaqMan＋SYBR Green Ⅰ组检测的HBV-DNA阳性血清均为阳性,其平均含量为10^8.55±0.32、10^5.79±0.29、10^3.81±0.30,与TaqMan组的10^8.49±0.31、10^5.69±0.34、10^3.72±0.26copies/ml0.320.290.300.310.300.25对应浓度值取10对数比较,无统计学意义（t=0.31、0.54和0.27,P〉0.05）;与SYBR Green I组的10^8.41±0.35,10^5.21±0.34和10^3.26±0.26copies/ml（不含未检出的两次血清）比较,除高浓度外,中低浓度有统计学意义（t=2.90和0.340.262.62,P〈0.05）。TaqMan＋SYBR Green I组和SYBR Green Ⅰ组阳性血清均出现明显熔解曲线,熔解温度（Tm）分别为71.8℃、72℃和79.8℃,阴性血清未出现扩增曲线和Tm值。结论SYBR Green Ⅰ联合TaqMan-PCR检测HBV-DNA时,具有能维持TaqMan-PCR的高灵敏度、特异性更强,并能同时检测HBV-DNATm的特点,为HBV的DNA多态性分析,尤其是在HBV基因分型方面提供了新的检测思路。     

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。     

Specific and sensitive diagnosis of syphilis using a real-time PCR for Treponema pallidum

A real-time PCR assay with a Taqman probe was developed that targeted the polA gene of Treponema pallidum. The test was validated using an analytical panel (n = 140) and a clinical panel of genital samples (n = 112) from patients attending a sexually transmitted infections clinic. High sensitivities and specificities of 94-100% were achieved using two real-time PCR platforms, the Rotor-Gene and the iCycler. The assay can be completed within 2 h, enabling reporting in <8 h. This fast and robust assay is suitable for implementation in routine laboratories for diagnosing primary syphilis.     

T cell receptor BV repertoires using real time PCR: a comparison of SYBR green and a dual-labelled HuTrec fluorescent probe

Real time PCR is a useful tool in immunological research but little has been published on the use of this technique in the measurement of T cell receptor (TCR) BV repertoires. We have compared the performance of SYBR green with that of a dual-labeled HuTrec (Human T cell receptor) fluorescent probe system. Serial dilutions of peripherals blood mononuclear cells were tested to compare the consistency of the two systems across multiple T cell receptor signal levels. Samples were diluted with non-TCR cDNA to simulate a low-level TCR signal within a tissue sample. The fluorogenic probe gave highly consistent results with a correlation coefficient of greater than 0.9 across a samples. SYBR green showed accurate results only when tested with high signal samples. Low-signal cDNA gave very poor results with SYBR green compared to the HuTrec fluorogenic probe with correlation coefficients as low as 0.65. Poorest performance occurred in the context of the simulated tissue sample with a high level of non-TCR DNA. Under these conditions, large amounts of nonspecific PCR product were generated which were detected by the SYBR green system and therefore distorted the results. SYBR green performed poorly when used with samples contaminated with significant quantities of non-target cDNA and samples with low target signal. It is therefore not an appropriate method for the measurement of TCR repertoires in small tissue samples. A dual-labeled HuTrec fluorescent probe produced a consistent TCR repertoire across a broad range of TCR signal levels and proved robust in the presence of contaminating non-TCR cDNA. We recommend the use of such a fluorescent probe in real time PCR for the assessment of TCR repertoires in small tissue samples. Where samples are assayed using SYBR green, agarose gel confirmation of PCR product specificity should be provided.     

X连锁性铁粒幼细胞贫血家系新发现ALAS2基因G514A突变

目的明确2例患有铁粒幼细胞贫血同胞兄弟系X连锁性铁粒幼细胞贫血,并鉴定其家系的ALAS2基因。方法利用PCR-SSCP技术分析该家系先证者,外祖父母,父母及其患病兄弟6人的ALAS2基因,确定存在基因突变,之后通过测序分析明确具体突变的位点及碱基。结果该两例患儿在ALAS2基因第5外显子都存在突变,该突变来自于母系家族,其母亲和外祖母为携带者;通过测序分析进一步明确具体的突变为ALAS2基因G514A突变,导致甲硫氨酸被异亮氨酸所替代。结论该家系患者为ALAS2基因突变所致的X连锁性铁粒幼细胞贫血,存在一种新的突变基因-ALAS2基因第5外显子G514A突变。     

Real-time PCR for the rapid detection of vanA,vanB and vanM genes

Background/PurposeTo evaluate the ability of quadruple Taqman probe real-time PCR to the detection of vanA, vanB and vanM in enterococcal isolates.MethodsA total of 343 strains, including 253 vancomycin-resistant enterococcus (VRE) strains and 90 non-VRE strains, were tested by both quadruple Taqman probe real-time PCR and gel-based PCR assay.ResultsWhen differentiating among three genotypes of vanA, vanB and vanM in VRE strains, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic accuracy and consistency of the quadruple Taqman probe real-time PCR were all 100%. Minimum.Inhibitory concentration (MIC) results showed that there was a wide MIC range of vancomycin and teicoplanin for the strains that harboring vanA/vanM gene respectively or harboring vanA and vanM genes simultaneously. However, the VRE strains with vanB genotype all were sensitive to teicoplanin.ConclusionConsidering the excellent PPV and low NPV, real-time PCR would be useful to monitor VRE-colonized or infected patients. However, further evaluation of the assay's performance in the clinical specimens is required, especially when considering that high level of PCR inhibitors present in these samples.