RB1基因突变的筛查技术和发病风险评估

RB1基因早已被确认为视网膜母细胞瘤的唯一致病基因,也是人类第一个被分离的肿瘤抑制基因。视网膜母细胞瘤（RB）是婴幼儿最常见的眼科肿瘤,也是一种致死性肿瘤。RB1基因的突变检测,对RB患者及家系成员的分子诊断、筛查、产前诊断、植入前遗传学诊断及早期治疗均具有重要意义。迄今为止,RB1基因中所发现的突变类型包括点突变、缺失、插入、易位、剪接突变以及各种复杂突变,几乎遍布基因的启动子和27个外显子,甚至还有部分内含子突变。但RB1基因不存在突变热点,这大大增加了RB1基因突变筛查的难度。RB1基因的突变不仅存在于视网膜母细胞瘤患者中,而且见于许多恶性肿瘤。本文对RB1基因突变筛查技术和发病风险评估的最新研究进展作一简单综述。     

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、基因表达、突变和多态性研究等多个领域。     

多重荧光定量PCR对mAchR1 表达相对定量分析

目的： 建立多重荧光通道定量PCR方法研究乙酰胆碱Ach对毒蕈碱受体 mAchRsⅠ型基因表达的影响。 方法： 分别用FAM和VIC不同荧光素标记的MGB-TaqMan探针同时对靶基因和看家基因的转录拷贝数进行多色多通道测定。结果: 多重荧光通道定量PCR与单通道荧光定量PCR比较没有差异。Ach作用SK-N-SH细胞24 h和72 h后mAchR1表达分别上调1.38倍和下降6.71倍(P<0.01)。 结论： MGB-TaqMan探针技术在多重荧光RT-qPCR技术中具有高灵敏度和高重复性。Ach对mAchR1 mRNA表达水平有调节作用，并有时间效应。     

实时荧光定量PCR检测常见性传播疾病病原体基因

目的;用实时荧光定量聚合酶链反应(FQ-PCR)技术准确定量检测常见性病病原体基因,为临床治疗提供依据.方法采用实时FQ-PCR技术,检测了临床送检标本3641份.结果淋球菌(NGH)1416份、沙眼衣原体(CT)1165份、解脲支原体(UU)765份,人乳头瘤病毒(HPV6.11)214份、梅毒螺旋体(TP)81份,阳性率分别为26%(368/1416)、27%(315/1165)、36.5%(279/765)、77%(165/214)、21%(17/81).阳性率差异有高度显著性(P＜0.001).NGH、CT、UU、HPV6.11、TP阳性标本DNA平均拷贝数2.3×106、8.5×105、2.4×104、5.6×107、3.8×105.146例治疗后复查,其转阴率为NGH63.9%(39)、CT70%(33)、UU44.7%(17).治疗后复查转阴率差异有显著性(P＜0.05),其DNA拷贝平均数有明显下降.结论实时FQ-PCR技术检测常见性病病原体基因,具有简便、快捷、特异性高、定量准确等优点,对其诊断和治疗有指导意义.     

双色竞争性荧光定量PCR检测唐氏综合征

目的 建立一种快速检测唐氏综合征(Down syndrome,DS)的双色竞争性荧光定量聚合酶链反应(dual-color competitive quantitative fluorescent polymerase chain reaction,DCC-QF-PCR)方法,并探讨其应用于DS产前诊断的可能性.方法 提取30例DS患者和60名正常人的外周血DNA,设计DSCR和USC2两基因的特异共用引物和双色特异性TaqMan探针,同一反应管中进行两基因的DCC-QF-PCR,并与DSCR和GAPDH两基因的QF-PCR实验结果进行比较.提取46份羊水胎儿细胞的DNA进行DSCR和USC2两基因的DCC-QF-PCR,并与染色体核型分析结果对比;克隆出DSCR和USC2的单克隆基因片段,定量后进行定量配比的DCC-QF-PCR,并计算DSCR∶USC2拷贝数的比值.结果 DCC-QF-PCR检测中,DS患者DSCR∶USC2拷贝数比值范围为1.41～1.74,显著高于正常人的0.93～1.15;而QF-PCR检测中,DSCR∶GAPDH拷贝数比值范围较DSCR∶USC2增大.46份羊水检测中,3份DSCR∶USC2拷贝数比值分别为1.61、1.64和1.54,为DS患儿,其余43份正常,与染色体核型分析结果一致;DSCR与USC2基因定量配比的DCC-QF-PCR所得DSCR∶USC2拷贝数比值范围与配比值差异无统计学意义(P＞0.05),检测结果较为稳定.结论 DCC-QF-PCR具有准确、快速、需模板量少和费用低廉等特点,该方法在DS的基因诊断和产前基因诊断中有较为广泛的应用前景.     

荧光定量PCR检测吉林地区NGU病原体基因

目的 用实时荧光定量聚合酶链反应(FQ-PCR）技术准确定量检测非淋菌性尿道炎(NGU)中解脲支原体(UU)、沙眼衣原体(CT)基因，进一步了解UU、CT感染情况，为临床治疗提供依据。方法 采用FQ—PCR技术检测标本2553份，其中UUl072份、CT1481份。结果 UU阳性率为39．4％(42／1072)，其中，男性阳性率为11．1％(40/360)，女性阳性率为53．7％(382/712)。CT阳性率为10．1％(149／1481)，其个，男性阳性率为10．6％(34／322)，女性阳性率为9．9％(115／1159)。UU感染率在男女性别之间有高度显著性差别(P＜0．01)，CT感染率在性别之间无显著性差别(P＜0．05)。在男性中UU与CT感染率之间无显著性差异(P＞0．05)。在女性CT与UU感染率之间有高度显著性羞异(P＜0．01)。结论 FQ-PCR技术检测UU、CT基因具有简便、快捷、特异性高、定量准确等特点，其结果对诊断、治疗有一定指导意义。     

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。     

用实时荧光定量PCR方法检测母血中的胎儿SRY基因

目的　从孕妇外周血浆中分离出胎儿DNA ,并加以鉴定 ,预防X连锁遗传病患儿的出生。方法　从孕早期、中期共 3 0 0名孕妇外周血浆中分离胎儿DNA ,用实时荧光定量聚合酶链反应 (fluorescencequantitativepolymerasechainreaction ,FQ PCR)的方法检测其中的Y性别决定区 (sex determiningregionY ,SRY)基因。结果　孕早期怀男胎的孕妇 82名 ,70名SRY基因阳性 ,其平均浓度为 ( 5 8.82± 2 0 .90 )拷贝 /ml,中位数为 5 8.5 0拷贝 /ml。孕中期怀男胎的孕妇 90名 ,SRY基因均为阳性 ,平均为 ( 15 2 .0 8± 62 61)拷贝 /ml,中位数为 14 9.3 5拷贝 /ml。怀女胎的孕妇均为阴性。结论　用实时荧光定量PCR的方法最早在孕42天的孕妇外周血浆中就可以检测到胎儿SRY基因 ,随孕周的增加 ,母血中胎儿DNA的量也在逐渐增加。实时荧光定量PCR技术在进行无创伤性产前性别诊断中有重要的价值。     

荧光定量聚合酶链反应技术在检测CT及UU中的应用

沙眼衣原体(Chlamydia trachomatis, CT)和解脲支原体(Ureaplasma urealyticum, UU)作为非淋菌性尿道炎(NGU)的主要病原体,在世界范围内,其流行和传播呈逐年上升趋势,在欧美等国家己超过了淋球菌,居于性传播疾病之首.如美国CT及UU引起的NGU发病数是淋菌性尿道炎(GU)的二倍[J].我国1993-1994年全国性病监测点报道,NGU的感染率仅次于淋病、尖锐湿疣,占第3位[2].而金氏报道,南京市1996年NGU的发病率为20.67%,己升至该地区性病发病率的第二位[3].CT及UU感染者一般临床症状不典型,且潜伏期也较长,因此易为临床所忽视,对CT和UU的诊断,主要依据实验室检测.本文应用荧光定量聚合酶链反应(FQ-PCR)技术,对682例疑为CT及UU感染的患者,进行了病原体基因的定量测定,就目前CT和UU的检验现状,作者还进行了简要评价,现报告如下.     

多重定量荧光PCR技术在Y染色体AZF微缺失检测中的应用研究

目的建立检测Y染色体无精子症因子（AZF）微缺失的多重定量荧光PCR体系。方法以5’FAM、JOE和TAMRA荧光基团标记PCR引物,建立包含Y染色体AZF4个亚区（AZFa～d）15个序列标签位点（STS）的多重定量荧光PCR体系;并对无精子症组、严重少精子症组及精液正常组进行Y染色体AZF微缺失检测。结果成功建立了检测Y染色体AZF微缺失的多重定量荧光PCR体系;200例男性中检测到Y染色体AZF微缺失16例,其中72例无精子症组7例,缺失率为9．7％,78例严重少精子症组9例,缺失率为15．4％,50例精液正常组未检测到缺失。无精子症组、严重少精子症组缺失率与精液正常组比较差异均有统计学意义（P〈0．05）。结论多重定量荧光PCR技术是一种快速、简便检测Y染色体AZF微缺失的方法,具有重要临床应用价值。     

先天性心脏病患儿22q11微缺失的定量荧光聚合酶链反应检测

目的 建立对22q11染色体微缺失进行快速批量检测的技术,用于非综合征先天性心脏病患 者染色体微缺失情况的检测.方法 采用定量荧光聚合酶链反应(quantitative fluorescent polymerase chain reaction,QF-PCR)法和8个短串联重复(short tandem repeat,STR)多态标记对79例中国汉族非综合征先天性心脏病(congenital heart defects,CHD)患者和84名正常对照者进行染色体22q11微缺失的检测.结果 缺失区域的STR标记在受检的非综合征型先天性心脏病患者中的平均杂合率为0.76,在正常对照人群中为0.79.在受检的1例法洛氏四联症患儿中检测到22q11微缺失(1.3%),经多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术验证结果正确.结论 采用QF-PCR法,可以高效批量地对22q11染色体微缺失进行筛查.     

多重定量连接酶链反应在遗传性耳聋检测中的应用研究

目的 建立以多重定量连接酶链反应(multiplex quantitative ligase chain reaction,MQ-LCR)为基础的聋病基因诊断技术,实现廉价、快捷、准确的检测目标.方法 针对GJB2基因230delC、299-300delAT,mtDNA A1555G,SLC26A4基因IVS7-2 A＞G、2168A＞G等5种突变类型设计相应的探针和引物,建立检测体系,从复旦大学儿科医院眼耳鼻喉咽科门诊中随机选择感音神经性聋儿98例以及听力正常对照30名,遵循双盲法原则分别以MQ-LCR法和DNA测序法进行检测,结果进行比较分析.结果 98例聋儿中发现聋病基因纯合或双突变48例,杂合突变31例,阴性19例,GJB2基因突变致病的占29.6%(29/98),mtDNAA1555G致聋的占4.08%(4/98),SLC26A4致聋的占15.31%(15/98).30名听力正常对照中发现杂合突变3例,阴性27例,其中GJB2基因235delC杂合突变2例,IVS7-2A＞G1例.经分析MQ-LCR和DNA测序法检测结果完全一致,未发现假阳性和假阴性.结论 所建立的MQ-LCR法检测聋病常见突变(235delC、299-300delAT、mtDNA A1555G、IVS7-2 A＞G、2168A＞G)具有价格便宜,操作快捷,结果准确可靠的特点,可应用于我国常见聋病基因的大规模筛查,为明确聋儿病因,降低聋生出生率提供一种很好的方法.     

Epidemiology of Epstein-Barr virus, cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay

A multiplex polymerase chain reaction (PCR) has been developed for the simultaneous detection of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Kaposi's sarcoma-associated herpesvirus (KSHV) in a clinical sample. Primers of multiplex PCR were designed to amplify specific regions of the EBV EBNA1, CMV IE2, and KSHV LANA genes. This multiplex PCR assay was found to have detection sensitivities of 1-10 copies of purified viral DNA cloned into the plasmid. To assess diagnostic and pre-clinical applications with this method, we utilized KSHV-positive primary effusion lymphoma (PEL) cells, EBV-positive Burkitt's lymphoma cells, CMV-infected fibroblast cells, and clinically prepared peripheral blood leukocytes (PBLs) that had been infected with viruses. We found that this multiplex PCR assay has high sensitivity and specificity for simultaneous detection of EBV, CMV, and KSHV genomes in a single amplification from a clinical material. Using this multiplex PCR assay, we investigated the prevalence of EBV, CMV, and KSHV in PBL samples from normal Japanese randomly selected. KSHV, EBV, and CMV genomes were detected in samples from 2 (0.2%), 377 (39.5%), and 27 (2.8%) of the 953 blood donors, respectively. Interestingly, both EBV and CMV genomes were detected in samples from all KSHV-positive donors.     

Rapid detection of novel BRCA1 rearrangements in high-risk breast-ovarian cancer families using multiplex PCR of short fluorescent fragments

Recent studies have revealed a significant proportion of BRCA1 exon deletions or duplications in breast-ovarian cancer families with high probability of BRCA1- or BRCA2-linked predisposition, in which mutations of these genes have not been found. The difficulty of detecting such heterozygous rearrangements has stimulated the development of several new screening methods. Quantitative fluorescent multiplex PCR is based on simultaneous amplification of multiple target sequences under conditions that allow rapid and reliable quantitative comparison of the fluorescence of each amplicon in test samples and in controls. The modified method described here, named quantitative multiplex PCR of short fluorescent fragments (QMPSF), is particularly well suited for large genes. All BRCA1 coding exons were analyzed using four multiplexes in 52 families without point mutations in the exons or splice-sites of BRCA1 and BRCA2, and selected because of high probability of a BRCA1- or BRCA2-linked genetic predisposition. Five distinct BRCA1 rearrangements were detected: a deletion of exons 8-13, a duplication of exons 3-8, a duplication of exons 18-20, a deletion of exons 15-16, and a deletion of exons 1-22-which is the largest deletion found so far within the BRCA1 gene. The method described here lends itself to rapid, sensitive, and cost-effective search of BRCA1 rearrangements and may be included into the routine molecular analysis of breast-ovarian cancer predispositions. Hum Mutat 20:218-226, 2002.     

Detection of Gallibacterium anatis by TaqMan fluorescent quantitative PCR

To better understand the prevalence of Gallibacterium anatis in different poultry species, a rapid and accurate method was developed to detect G. anatis using a TaqMan fluorescent quantitative polymerase chain reaction (qPCR). Specific primers and a TaqMan probe were designed based on the reference gtxA gene sequence. The qPCR standard curve showed a good linear relationship, and the method showed good reproducibility, sensitivity, and specificity, indicating its suitability for G. anatis identification and quantitative analysis. A comparison of the detection results in 160 clinical swab samples showed that the detection rate (54.4%) of the qPCR for G. anatis was better than that of two conventional methods: gyrB gene-based qPCR for G. anatis (51.9%) and culture-based identification (34.4%). G. anatis was detected in layer chicken (77.3%), Silkie chicken (72.7%), and duck (27.1%) with relatively high detection rates, whereas dove (8.8%) and quail (3.0%) showed lower detection rates, indicating the different prevalence of G. anatis in different fowl species.     

实时荧光定量PCR检测儿童下呼吸道感染的WU多瘤病毒

目的 建立并应用检测儿童下呼吸道感染WU多瘤病毒(WU polyomavirus)的实时荧光定量PCR( real-time fluorescent quantitative PCR,FQ-PCR)方法.方法 选择WU多瘤病毒的VP2基因作为检测的目标基因,设计FQ-PCR引物和检测探针,以重组质粒为标准品建立标准曲线,并对该方法的特异性、灵敏度、重复性进行评价;应用该方法对温州医学院附属温岭医院收集的临床下呼吸道感染患儿的痰液、咽拭子846份及血清标本846份进行定量检测.结果 本研究建立FQ-PCR检测方法,质粒标准曲线的方差系数为0.998,灵敏度可达到50拷贝;应用该方法检测700份痰液标本,检测到7例阳性标本,146份咽试子标本中未检测到阳性标本,总阳性率为1.00％ (7/700),846份血清标本未检测到阳性标本.结论 本研究建立的FQ-PCR方法可以特异、快速、灵敏地对儿童下呼吸道感染的WU多瘤病毒进行定量检测;痰液标本较咽拭子或血清标本更适用于WU多瘤病毒感染的核酸检测.     

孕妇外周血中胎儿短串联重复序列基因型的检测

目的探讨利用孕妇血浆中胎儿DNA进行遗传病产前基因诊断的可行性。方法应用9个具有高度多态性的短串联重复序列（short tandem repeat，STR）位点，以多重荧光PCR方法对10名健康孕妇孕早、中、晚期共30份血浆标本和非孕期血浆标本中DNA进行STR等位基因扩增。同时检测孕妇丈夫的外周血本，然后进行基因扫描及对照分析，判断胎儿父源性等位基因在孕妇血浆中的检出情况。结果10名孕妇均足月分娩，男婴3名，女婴7名。10名孕妇血浆中均检出胎儿父源性等位基因，即胎儿DNA。30份孕期血浆标本中有23份检出胎儿DNA，其中孕早期标本6份（6／10）；孕中期标本8份（8／10）；孕晚期标本9份（9／10）。7份标本未见到胎儿DNA。结论应用多重荧光PCR方法对孕妇血浆中DNA进行SFR多态化点的复合扩增，可获得男性和女性胎儿DNA信息，可用于无创伤性产前诊断。     

Rapid determination of zygosity and common aneuploidies from amniotic fluid cells using quantitative fluorescent polymerase chain reaction following genetic amniocentesis in multiple pregnancies

Following second-trimester twin amniocentesis, we used quantitative fluorescent polymerase chain reaction (QF-PCR) assays and polymorphic small tandem repeats (STR) for rapid determination of zygosity and common aneuploidies from amniotic fluid (AF) cells in four pregnancies with like-sex twins, fused placentae and inconclusive chorionicity. The first and the second cases were suspected to have inadvertent sampling of the same amniotic cavity twice. The first case showed a dizygotic (DZ) pattern and repeat amniocentesis was thus avoided. The second case was monozygotic (MZ) and was complicated by discordant fetal growth and twin-twin transfusion syndrome. The third case was associated with a co-twin malformation, occipital encephalocele. DNA studies revealed MZ twinning with a discordant structural defect. The fourth case was associated with co-twin abnormalities of cystic hygroma and hydrops fetalis. DNA studies showed DZ twinning with discordant structural and chromosomal defects. The QF-PCR assay with STR has the advantages of rapid determination of zygosity and common aneuploidies in AF cells. This simple test appears to be useful in the instances of possible inadvertent puncture of the same amniotic cavity twice during amniocentesis and of discordant fetal structural and/or chromosomal abnormalities following genetic amniocentesis in multiple pregnancies with uncertain chorionicity.