Real-time PCR的研究进展及其应用

本文综述了real-time PCR的原理、定量方法及其在各领域的应用。     

双链置换探针实时PCR用于DNA池的等位基因频率测定

结合荧光双链置换探针的特异性和实时PCR的准确定量能力,建立了一种新颖、准确、价廉且高通量的测定DNA池等位基因频率的方法.该方法采用不同荧光标记的双链置换探针1次PCR反应即可测定出等位基因频率.实验以β 地中海贫血CDs41 42( TCTT)突变为对象,分别用荧光染料FAM和ROX标记野生型和突变型探针,由实时PCR检测建立的等位基因浓度与循环阈值的响应曲线计算DNA池的等位基因频率.结果表明,建立的系列等位基因浓度与循环阈值呈线性关系,线性相关系数分别为0.9977(野生型等位基因)和0.9938(突变型等位基因),可检测的最低等位基因频率达1%,等位基因频率在1%～90%范围内测定误差小于4%.该方法可广泛用于流行病学调查、遗传连锁分析以及全基因组连锁不平衡扫描等领域.     

虫荧光素酶N末端缺失

目的是通过虫荧光素酶Ｎ末端氨基酸的缺失,研究缺失对酶活性的影响。采用聚合酶链式反应的方法,构建虫荧光素酶Ｎ－末端缺失７,８,９个氨基酸残基的突变体,导入大肠杆菌ＤＨ５α菌株中直接得到表达,再分离纯化粗酶,并检测酶活。结果表明,Ｎ末端缺失７个氨基酸的突变体几乎没有活性（小于天然活性的０．５％）,Ｎ末端缺失８个以上氨基酸残基的突变体则完全丧失活性。由于Ｎ末端缺失６个氨基酸的突变体保持了７７％的酶活性,因而萤火虫荧光素酶Ｎ末端第７个氨基酸与酶的催化活性密切相关。     

Cloning and expression of the vp39 gene ofBombyx mori nuclear polyhedrosis virus inE. coli

The nuclear capsid protein gene (vp39) ofBombyx mori nuclear polyhedrosis virus (BmNPV) was amplified successfully by PCR technique and inserted into pGEM 3zf(+). The 5′ and 3′ terminal area of the amplified vp39 gene were sequenced with silver-staining dideoxy method. Bmvp39 gene was sub-cloned into the expression vector pRSET-A, and transformed intoE. coli BL21. This gene was highly expressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 38 kd, and the expressed amount reached maxium in 4 h with IPTG induction. Supported by the National Natural science Foundation of China and the Doctoral Foundation of Edn carto Committee Liu Deli: born in 1954, Doctoral Candidate from Huazhong Normal University To whom correspondence should be addressed: (027-7882712-2938)     

雏鹅新型病毒性肠炎病原快速检测技术的建立及应用

根据小鹅瘟病毒(GPV)和鹅源禽腺病毒(FAV)基因组序列特征分别设计了各自的PCR引物,经对反应条件的优化建立了能同时检测两种病毒的二重PCR方法．该法能从雏鹅新型病毒性肠炎和小鹅瘟疫苗株以及分离的鹅源腺病毒蚀斑克隆株分别扩增出相应的特异性产物,而对其它常见鹅病的病原及正常鹅胚肝组织的扩增结果均为阴性;对来自广西不同地方的36份临床病例样品的检测发现,其中具有雏鹅新型病毒性肠炎典型病变的22份样品均可检出GPV和FAV两种病毒．其它的有4份只检出GPV,3份只检出FAV;12份健康雏鹅肝组织中仅有一份检测到GPV;健康成鹅泄殖腔中GPV和FAV的检出率分别达44．29％、37．14％二者都有的占1．43％．结果表明,雏鹅新型病毒性肠炎的病原可能包括GPV和FAV两种病毒;建立的PCR诊断技术具有快速、敏感、特异的特点,可用于该病征的临床快速鉴别诊断以及流行病学的调查研究．     

垃圾渗滤液处理系统中微生物群落结构变化研究

采用基于PCR的变性梯度凝胶电泳(PCR-DGGE) 和 real-time PCR 方法, 分析了深圳市某垃圾渗滤液处理系统中总细菌和硝化细菌的生物量及群落结构变化, 探究生物量与水质变化间的关系及环境因素对微生物群落结构的影响。结果表明, 该垃圾渗滤液处理系统具有很好的氨氮和总有机碳(TOC) 去除效果, 总去除率分别为 99.9% 和 91.4% 。在垃圾渗滤液处理系统中, 总细菌、氨氧化菌(AOB) 、亚硝酸盐氧化菌(NOB) 的生物量与 TOC 及氨氮浓度没有显著相关性(p > 0. 05); 溶解氧不影响AOB 生物量, 但明显影响NOB 生物量。微生物群落结构及多维尺度(DMS) 分析表明:在该废水处理系统中, DO浓度对总细菌和 NOB 微生物群落结构有重要影响; 而氨氮浓度是影响 AOB 群落结构的关键因素。     

石蒜单染色体的显微分离及体外扩增

建立了石蒜单染色体的分离及体外扩增的方法。石蒜根尖经卡诺固定液固定后，再用果胶酶和纤维素酶酶解处理，制得标本，在倒置显微镜下，用自制的毛细管针挑取目的的染色体。将分离的石蒜染色体放入0．2mLEppedorf管中，经蛋白酶K处理后，进行DOP－PCR扩增，获得DNA片段。琼脂糖凝胶电泳显示扩增产物的长度大约为100－5000bp。此法为构建石蒜单染色体基因组库和筛选其特异性探针奠定了基础。     

本生烟组织培养及遗传转化体系的建立

本文研究了通过农杆菌侵染获得本生烟(Nicotiana benthamiana)转基因植株的方法。将本生烟中上部叶片消毒后切成5mm×5mm左右的小块作为外植体,在含有6-BA(3mg/L)和NAA(0.2mg/L)的MS培养基中培养2~3d,用含有表达载体的农杆菌侵染后共培养3~4d,转入含有6-BA(3mg/L)、NAA(0.2mg/L)和卡那霉素(100mg/L)的MS培养基中培养2~3周。将外植体边缘产生的愈伤组织和芽点转移到只含有卡那霉素(100mg/L)的MS培养基中继续培养,2周后分化出大量不定芽。继续培养2周,当芽长1~3cm时,将芽转移到含IBA(0.1mg/L)的MS培养基中分化生根,得到再生植株。当再生植株高8~10cm、根长4~5cm以上时,移栽到经过灭菌的营养土中。通过实时荧光PCR扩增,从获得的植株中检测35S启动子和NOS终止子外源基因片段,判定所得的植株的确为转基因植株。最后对本生烟组织培养中应该注意的问题进行了讨论。     

MGB probe assay for rapid detection of mtDNAl1778 mutation in the Chinese LHON patients by real-time PCR

Objective： Leber＇s hereditary optic neuropathy （LHON） is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA （mtDNA）. Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder （MGB） probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction （PCR）. Methods： Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation, with 20 normal individuals as a control group at the same time. A real-time PCR involving two MGB probes was used to detect the mtDNA 1 1778 mutation and heteroplasmy. A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones. Results： All 48 LHON patients and their maternal relatives were positive for rntDNA11778 mutation in our assay, 27 heteroplasmic and 21 homoplasmic. Eighteen cases did not show an occurrence of the disease, while 9 developed the disease among the 27 heteroplasmic mutation cases. Eleven did not show an occurrence of the disease, while 10 cases developed the disease among 21 homoplasmic mutation cases. There was a significant difference in the incidence between the heteroplasmic and the homoplasmic mutation types. The time needed for running a real-time PCR assay was only 80 min. Conclusion： This real-time PCR assay is a rapid, reliable method for mtDNA mutation detection as well as heteroplasmy quantification. Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.