荧光定量PCR技术及其应用研究进展

荧光定量PCR是近年发展起来的一种新的实时定量检测特定核酸技术,它是核酸探针技术、荧光共振能量传递技术和PCR技术的有机结合,而荧光探针是荧光定量PCR的核心.荧光定量PCR具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应争特点,是对原有PCR技术的革新,扩大了PCR的应用范围.论文综述了FQ-PCR技术的原理、FQ-PCR实时定量检测系统及其应用.     

荧光定量PCR技术及其在动物传染病定量检测中的应用

荧光定量PCR是近年发展起来的一 种新的实时定量检测特定核酸技术,它是核 酸探针技术、荧光共振能量传递技术和PCR 技术的有机结合,而荧光探针是荧光定量 PCR的核心。目前报道的荧光探针主要有 TaqMan、Amplisensor、分子信标、Lightcycler 以及在这4种探针基础上发展起来的其他荧 光探针。各种荧光探针在定量PCR中的作 用是识别和报告特定核酸。这些荧光探针与 相应的靶分子杂交时经历一个自发荧光形成 或消失的构象变化,只有与完全互补的靶核 酸杂交时才会出现这种变化,当靶核酸存在 碱基错配或缺失时,荧光探针就不会与之杂 交,也不会出现荧光的变化。在检测时根据 这种荧光变化来确定检测中特定核酸的存在 和量的多少。荧光探针用于定量PCR不但 提高了PCR的检测灵敏度,还能在同一封闭 管中对模板进行准确定量检测,特别适合特 定核酸扩增的实时监测。荧光定量PCR用 于动物传染病的诊断,不但能定量检测病原 体感染的强弱和在机体内的分布,还具有灵 敏、快速、省力的特点。     

TaqMan MGB探针实时荧光定量PCR快速检测牛鹦鹉嗜热衣原体

为了建立特异、敏感、快速检测鹦鹉热嗜衣原体的TaqManMGB探针实时荧光定量PCR方法,针对支原体ompA基因的保守区设计特异性引物和探针,建立鹦鹉热嗜衣原体TaqManMGB探针实时荧光定量PCR检测方法,验证方法的特异性、敏感性和稳定性。并对来自宁夏地区三个规模化奶牛养殖场的376份流产奶牛样品利用鹦鹉热嗜衣原体IHA诊断试剂盒和TaqManMGB探针实时荧光定量PCR进行检测。结果表明：建立的TaqManMGB探针实时荧光定量PCR方法敏感性为10培的总DNA,是一种可靠、快速、灵敏的检测鹦鹉热嗜衣原体的方法,并且成功应用于奶牛鹦鹉热嗜衣原体样本的检测。     

TaqMan MGB探针实时荧光定量PCR快速检测牛鹦鹉嗜热衣原体

为了建立特异、敏感、快速检测鹦鹉热嗜衣原体的TaqMan MGB探针实时荧光定量PCR方法,针对支原体ompA基因的保守区设计特异性引物和探针,建立鹦鹉热嗜衣原体TaqMan MGB探针实时荧光定量PCR检测方法,验证方法的特异性、敏感性和稳定性。并对来自宁夏地区三个规模化奶牛养殖场的376份流产奶牛样品利用鹦鹉热嗜衣原体IHA诊断试剂盒和TaqMan MGB探针实时荧光定量PCR进行检测。结果表明:建立的TaqMan MGB探针实时荧光定量PCR方法敏感性为10 fg的总DNA,是一种可靠、快速、灵敏的检测鹦鹉热嗜衣原体的方法,并且成功应用于奶牛鹦鹉热嗜衣原体样本的检测。     

高致病性猪繁殖与呼吸综合征病毒实时荧光定量PCR检测方法的建立

<正>根据GenBank公布的24株高致病性猪繁殖与呼吸综合征病毒毒株和5株PRRSV经典毒株的保守区基因序列,使用PrimerExpress3.0软件设计并合成实时荧光定量PCR(Real-timeFluorescent Quantitative PCR,Real-time FQ-PCR)用引物和探针,建立了Real-time FQ-PCR检测方法以鉴别检测高致病性猪繁殖与呼吸综     

实时荧光定量PCR技术及在猪重大疫病中的诊断应用

实时荧光定量PCR技术（real-time fluorescent quantitative PCR．FQ-PCR）是在PCR反应体系中加入荧光染料或荧光探针,利用荧光信号积累实时监测整个PCR反应进程,最后通过标准曲线对未知模板进行定量分析。该技术具有操作简便、特异性强、灵敏度高、重复性好、定量准确、快速高效、全封闭...     

实时荧光定量PCR技术及应用

实时荧光定量PCR技术是在常规PCR基础上发展起来的核酸定量技术,于1996年由美国Applied Biosystems公司推出.当前,实时荧光定量PCR技术应用范围很广,例如,能从粪便材料中分离出的细菌DNA来精确定量检测肠道菌,使我们能详细地分析肠道中优势菌与荧光原位杂交(FISH)及培养方法检测不到的非优势菌,为理解肠道菌群与机体状况关系提供重要线索.该技术从原理上分为荧光染料检测模式和荧光探针检测模式两方面.     

实时定量PCR技术在弓形虫研究中的应用

实时定量PCR技术（real-time polymerase chain reaction/quantitative real time polymerase chain reaction,real-time PCR/qPCR）是在PCR反应体系中加入荧光基团,利用荧光信号积累实时监测整个PCR进程,最后通过标准曲线对未知模板进行定量分析的方法;该技术具有特异性强、灵敏度高、定量准确和快速等优点,在生物医学领域中得到广泛的应用。笔者对实时定量PCR技术的原理和近几年新兴的荧光探针的原理和类型进行了论述,并对实时定量PCR技术在弓形虫基础生物学、诊断及检测、药物和疫苗研究中的应用进行了综述。     

弓形虫Real-time PCR检测方法的建立和初步应用

根据已发表的弓形虫529 bp高拷贝序列,设计特异性引物和探针,建立TaqMan探针法的实时荧光定量PCR检测方法。将该方法用于360份临床样本的检测,并与常规PCR和环介导等温扩增(LAMP)技术进行对比分析。结果表明:Real-time PCR检测的阳性率(11.11%)高于LAMP(7.5%)和常规PCR(3.61%)。采用本实验室建立的Real-time PCR,对收集于不同地区4种动物的750份临床样本检测发现,不同种类动物血液样本中均可检测到弓形虫感染,其中羊的感染率最高(17.8%),猪次之(4.22%),牛较低(1.98%),犬最低(1.37%)。     

实时荧光PCR技术在动物疫病诊断中的应用研究

实时荧光定量PCR技术于1996年美国Applied Biosystems公司推出以来,取得飞速发展。该技术通过在普通PCR反应体系中加入荧光探针或荧光染料,在PCR过程中直接检测荧光信号的变化情况,从而能够实时监测整个PCR进程,     

Development of Real-time Quantitative PCR Method for Rapid Detection of Enterohemorrhagic Escherichia coli O157∶H7

A Real-time quantitative PCR method was developed for the rapid detection of enterohemorrhagic Escherichia coli （EHEC） O157∶H7 based on the primers and TaqMan probes which were designed for the conservative domain of rfbE gene of EHEC O157∶H7.The results showed that the sensitivity of Real-time PCR was 7.3 CFU/mL.20 from 310 samples of meat,egg,milk and its products,animal diarrhea materials and artificial contamination samples were positive detected by Real-time PCR assay,which was in accordance with the testing result according to AOAC standard.The results indicated that Real-time PCR assay was a sensitive,rapid and simple tool.     

伪狂犬病病毒野毒株与gE基因缺失疫苗株TaqMan实时荧光定量PCR鉴别方法的建立

为实现对伪狂犬病病毒（pseudorabies virus,PRV）野毒株与gE基因缺失疫苗株的快速、敏感、特异的鉴别诊断,本试验针对PRV gD和gE基因设计了2套特异性引物和TaqMan探针,建立了PRV野毒株与gE基因缺失疫苗株的TaqMan实时荧光定量PCR鉴别方法,对引物和探针浓度、退火温度等进行了优化,对方法进行敏感性、特异性、重复性试验,并进行临床样品检测。结果显示,建立的针对gD、gE基因的TaqMan实时荧光定量PCR方法线性相关系数（R²）分别为0.996和0.980,均呈良好的线性关系;检测限分别为39.4和12.1拷贝/μL;与圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒均无交叉反应;重复性试验结果显示,针对gD基因的批内和批间变异系数分别为1.43%~1.86%、1.10%~2.07%,针对gE基因的批内和批间变异系数分别为0.98%~1.41%、1.12%~1.86%。应用建立的TaqMan实时荧光定量PCR与普通PCR分别对11份临床疑似感染样品进行检测,阳性率分别为36.4%和27.3%。结果表明,该方法敏感性高、特异性强、重复性好,可作为伪狂犬病病毒野毒株与gE基因缺失疫苗株的早期鉴别诊断和定量检测的有效手段。     

Q热贝纳柯克斯体TaqMan荧光定量PCR检测方法的建立

根据Q热贝纳柯克斯体(Coxiella burnetii)插入IS1111序列设计引物和探针,建立快速检测Q热的TaqMan实时荧光定量PCR方法。以梯度稀释含有目的扩增片段的重组质粒作为标准品,进行定量PCR反应。结果显示,该方法能够检测出10个拷贝数的阳性质粒;标准曲线相关系数为0.995,扩增效率为103%;结核分枝杆菌(M.tuberculosis)、衣原体(C.psittaci)、布鲁氏菌(Brucella.spp)及牛血液的核酸样本特异性检测结果均为阴性。本研究建立的TaqMan荧光定量PCR法灵敏度高、特异性好,对Q热的检测与鉴定中具有良好的应用前景。     

Establishment and Application of A Double TaqMan MGB Real-time PCR Assay for Detection of Canine Distemper Virus and Canine Parvovirus

This study was aimed to establish a double TaqMan MGB Real-time PCR assay to simultaneously and specifically detect canine distemper virus (CDV) and canine parvovirus (CPV) in one reaction.Two pairs of specific primers for CDV and CPV,along with two TaqMan MGB probes for each virus were designed in the assay basing on CDV H gene and CPV VP2 gene sequences.The specificity,sensitivity and repetition of the double TaqMan MGB Real-time PCR assay were tested,and 48 samples taken from clinic suspicious CDV and CPV infected canines had been testified by the established double TaqMan MGB Real-time PCR.The results indicated that the doulde TaqMan MGB Real-time PCR assay was successfully established,and the number of standard curve correlation (R²) of CDV and CPV were 0.997 and 0.993,respectively.The specificity of the double TaqMan MGB Real-time PCR assay revealed that amplifications were showed on CDV and CPV samples,but other pathogens and negative controls had no amplifications;The sensitivity of CDV and CPV were both 10 copies/μL.Meanwhile,14 CDV positive samples,19 CPV positive samples and 4 CDV/CPV double positive samples were detected,which were consistent with the results of the sequencing.Therefore,the established double TaqMan MGB Real-time PCR assay had high sensitivity,specificity and flux accurate quantitative,which could be applied to clinical CDV/CPV infection each periods.     

鸭坦布苏病毒和鸭瘟病毒二重荧光定量RT-PCR方法的建立

The study was conducted to establish the duplex Real-time PCR assay for detecting both duck tembusu virus (DTMUV) and duck plague virus (DPV). According to the sequences of DTMUV E gene and DPV UL6 gene in GenBank, two sets of specific oligonucleotide primers for DTMUV and DPV along with two TaqMan probes were designed. The duplex Real-time PCR assay was developed through optimization of reaction conditions and validation of specificity, sensitivity and repetitiveness of the method. The sensitivity of the assay were both 100 template copies for DTMUV and DPV. There was no specific bands of the same sizes were amplified from other duck pathogens, such as duck Newcastle disease virus, duck hepatitis virus, muscovy duck parvovirus, duck circovirus, H9 subtype avian influenza virus, egg drop syndrome virus. This duplex Real-time RT-PCR assay is a sensitive, quick, specific and quantitative test for detection of DTMUV and DPV, and will be useful for the control of these viruses in ducks.     

鸽白细胞介素8基因实时荧光定量PCR检测方法的建立

为建立鸽白细胞介素8（interleukin-8,IL-8）基因实时荧光定量PCR检测方法,本研究根据GenBank上公布的鸽IL-8基因序列,在保守区域设计1对特异性引物,以鸽子淋巴细胞提取的核酸为模板扩增鸽IL-8基因部分片段并克隆到pMD18-T载体上。提取重组质粒通过系列制备标准品,建立鸽IL-8基因SYBR GreenⅠ染料法实时荧光定量PCR标准曲线,并进行特异性、敏感性和重复性试验。结果显示,实时荧光定量PCR熔解曲线呈单一熔解峰,试验线性相关系数为1.0,IL-8基因的扩增效率为103%。敏感性结果显示最低可检测到16个拷贝数;用该方法检测其他鸽白细胞介素细胞因子（IL-1β、IL-6、IL-18）和双蒸水的结果均为阴性。批间、批内变异系数均≤1.52%。因此,本研究建立的实时荧光定量PCR检测方法可用于鸽IL-8 mRNA的检测,为病毒感染宿主细胞后细胞因子表达的定量分析奠定基础。     

Development of a Quantitative Real-time PCR Method for Detection of Pigeon IL-8 Gene

To develop a quantitative Real-time PCR method for detection of pigeon interleukin-8 (IL-8),a pair of specific primers was designed based on the conserved region of IL-8 gene sequence published on GenBank.A fragment of IL-8 gene was amplified from the pigeon lymphocytes template and cloned into pMD18-T vector.Plasmid DNA was extracted from the bacteria and was serially diluted to serve as a standard.A standard curve for the SYBR Green Ⅰ of quantitative Real-time PCR was established and the specificity,sensitivity and reproducibility of this assay were investigated.The results showed that the quantitative Real-time PCR melting curve only had one single melting peak.The assay was linear with R² values was 1.0;The reaction efficiency for the pigeon IL-8 gene was 103%.The detection limit of this assay was 16 copies per reaction.Other interleukin including IL-1β,IL-6 and IL-18 and double distilled water control were tested by this assay and the results were all negative.The CV values of intra- and inter-assay were less than 1.52%.The established quantitative Real-time PCR assay of this study was suitable for the detection of pigeon IL-8.It would provide basis for analyzing cytokine expression quantitatively after the host cell was infected by the virus.