Real-time fluorescent quantitative PCR非特异性扩增的研究进展

Real-time fluorescent quantitative PCR(RT-qPCR)因其高效快速、操作简便、高度敏感等优点获得广泛应用,但因其强大的放大功能而容易出现非特异性扩增的问题,尤其是荧光染料法中更加明显。文章对RT-qPCR的原理、非特异性扩增的发生因素、解决方案及该技术的应用前景进行了综述。

Application of Real-time Fluorescent Quantitative PCR in Plant

Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.

Detection of Lactobacillus acidophilus in Fermented Material by Real-time Fluorescent Quantitative PCR

The species distinctive PCR primer of Lactobacillus acidophilus （ L. acidophilus） was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative PCR （RT-PCR）. Analysis on RT-PCR results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-PCR method in the test. indicating that the established RT-PCR method could be aookued to the detection of L. acidophilus in fermented material.

Application of Real-time Fluorescent Quantitative PCR in Studies on Plants

Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.

Establishment of a Real-time Fluorescent Quantitative PCR Assay for Detection of Genetically Modified Maize Line MON88017

In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value （ 1. 541% ） was close to the actual value （ 1.50% ） and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.

Detection of Ratoon Stunting Disease in Virus-free Seedcane via Real-time Fluorescence Quantitative PCR

This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease （RSD） in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from six plants of sugarcane ROC22, which had been confirmed RSD-positive by detecting the sugarcane juice, by employing the sugarcane seedlings production protocol. Real-time fluorescence quantitative PCR was used to detect RSD pathogens in tissue culture sam- pies. The results showed that target fragment of RSD pathogens was not found in all 10 samples in real-time fluorescence quantitative PCR, with the Ct values of 37 - 39. The healthy tissue culture sugarcane seedlings do not carry RSD pathogens, indicating that adopting healthy seedcane seedlings production technique could thoroughly get rid of RSD pathogens.

Development and Preliminary Application of SYBR Green I Real-Time Fluorescence Quantitative PCR Method for Detecting Porcine Parvovirus Virus

According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 （NC001718） available in GenBank （NC_001718）, a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.

Primary application of a real-time quantitative polymerase chain reaction for the detection of human breast cancer related novel gene-Metadherin expression

Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.

Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction

Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.

Development of real-time PCR method for rapid detection and quantification of Heterosigma akashiwo

To rapidly detect the harmful algae H.akashiwo qualitatively and quantitatively, sequences of the 18S rDNA deduced from H.akashiwo were used for designing species-specific primers, and a RFQ-PCR (Real-time Fluorescent Quantitative Polymerase Chain Reaction) method was developed for quantitative detection of H.akashiwo. Primer H.akashiwo and TaqMan probe were designed, and the specificity of primer was checked with PCR. A calibration curve was constructed with cycle threshold value against visual counted cell number. And the value of the curve was tested with other H.akashiwo samples, which were assayed with both the RFQ-PCR method and visual count under microscope.

基于PMA-qPCR检测青枯菌5号生理小种活菌的方法

青枯菌5号生理小种(Ralstonia solanacearum race 5)是引发桑青枯病的病原菌。为了改进常规PCR方法不能对活体病原菌进行准确鉴别和定量分析的不足,建立叠氮溴化丙锭(PMA)预处理结合荧光定量PCR(q PCR)检测青枯菌5号生理小种活菌的方法。该方法首先将待检测样品的病原菌细胞用质量浓度为15 ng/μL的PMA暗孵育10 min后,在卤钨灯下曝光5min,以消除死菌细胞DNA的PCR扩增信号。用PMA-q PCR方法对含青枯菌5号生理小种活菌和死菌的混合样本的检测结果表明,死菌的存在不会对活菌细胞DNA的扩增产生影响。建立的PMA-q PCR标准曲线显示,在菌液浓度为103~107CFU/m L的范围内,qPCR循环阈值(Ct)与菌液浓度的对数值之间呈良好的线性关系,线性方程为y=-3.477x+47.489,R^2=0.997。建立的PMA-q PCR方法能更为准确地检测复杂样本中的青枯菌5号生理小种活菌数量,有助于对桑树青枯病的早期诊断和及时制定病害防控措施。

不同诱导条件下的VBNC状态副溶血弧菌的PMA-qPCR定量检测及其呼吸活性分析

定量检测不同诱导条件下的活的非可培养(viable but non-culturable,VBNC)状态副溶血弧菌的变化情况,并对VBNC菌的呼吸活性进行分析。采用叠氮溴化丙锭(propidium monoazide,PMA)与荧光定量聚合酶链式反应(fluorescence quantitative polymerase chain reaction,q PCR)结合的技术(PMA-q PCR)定量检测副溶血弧菌活菌数。结合激光扫描共聚焦显微镜和流式细胞仪优化传统CTC(5-cyano-2,3-ditolyl tetrazolium chloride)呼吸检测法对VBNC状态的副溶血弧菌进行分析。结果表明PMA-q PCR检测技术结合平板计数的方法,可用于定量检测副溶血弧菌诱导过程中活菌数和可培养菌数的变化情况,并利用差值测定出VBNC细胞的浓度。在不同的温度、Na Cl含量和氯霉素含量的诱导条件下,副溶血弧菌在其中的7种条件下在60 d内进入了VBNC状态。VBNC细胞终浓度最低为5.75×10~2 CFU/m L,最高为1.70×10~5 CFU/m L。在细胞呼吸实验中,采用CTC与4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole,DAPI)双染法能在激光扫描共聚焦显微镜下清晰地观察到VBNC细胞表面的红色荧光,有效区分死活细胞。同时利用流式细胞仪能够根据荧光强度快速区分呼吸活性呈阴性和阳性的细胞。本研究为VBNC细菌的检测和生理特性的研究提供了新的思路和依据。

常规PCR与实时荧光定量PCR(qPCR)技术敏感性比较

对70匹采自青海省海南藏族自治州兴海县和贵南县喜马拉雅旱獭体表寄生蚤2科3属3种,包括斧形盖蚤(Callopsylla dolabris)、谢氏山蚤(Oropsyllasilantiewi)、人蚤(Pulex irritans)进行巴尔通体检测,并对11个阳性样品进行常规PCR和实时荧光定量PCR(qPCR)的敏感性比较。通过常规PCR反应产物和qPCR反应产物琼脂糖凝胶电泳分析,发现qPCR检测敏感性高于常规PCR。此项技术的应用将对巴尔通体感染的早期快速诊断、监测和流行病学调查等研究提供有效方法。

芸薹根肿菌活细胞PMAxx-qPCR快速定量检测方法的建立与应用

【目的】由芸薹根肿菌(Plasmodiophora brassicae)侵染引起的十字花科根肿病是一种世界性土传病害,病原菌长期存在于土壤中,对十字花科作物造成严重威胁。改良叠氮溴化丙锭(propidium monoazide xx,PMAxx)可选择性地穿透受损的死细胞膜,并抑制死细胞DNA的实时荧光定量PCR(qPCR)扩增。本文将PMAxx与qPCR技术相结合,建立一种快速检测芸薹根肿菌活菌的方法,为根肿病的早期诊断及制定科学的防控措施提供依据。【方法】配置浓度分别为0、5、10、20、40、60μmol·L^(-1)的叠氮溴化丙锭PMA和改良叠氮溴化丙锭PMAxx,比较两种核酸染料对芸薹根肿菌死细胞DNA扩增的抑制效果,确定最佳核酸染料及工作浓度;设置光照时间分别为0、2、5、10、15和20 min,进行最佳光照时间的优化,建立芸薹根肿菌活细胞PMAxx-qPCR快速检测体系。设置芸薹根肿菌活孢子百分比为0、0.01%、0.1%、1%、10%、25%、50%、75%和100%的混合体系,验证PMAxx-qPCR体系的准确性,并应用于田间土壤样本中芸薹根肿菌活孢子的定量检测。【结果】PMAxx对芸薹根肿菌死细胞DNA的扩增抑制效果更好,当芸薹根肿菌浓度为1×10^(8)个孢子/mL,PMAxx预处理的最适终浓度为4μmol·L^(-1),最佳光照时间为10 min时,可有效地抑制死孢子DNA的扩增,仅以有活力孢子DNA为靶标选择性地扩增。利用PMAxx-qPCR技术检测已知不同活孢子比例的菌悬液样品,各样品实测孢子存活率和理论存活率之间呈正相关(R^(2)=0.992)。对田间采集的25份土壤样本,采用PMAxx-qPCR方法检测到11份样本中携带芸薹根肿菌,活细胞DNA浓度为32.35-6.97×10^(3)fg·g^(-1)。【结论】建立了基于PMAxx-qPCR的芸薹根肿菌活细胞定量检测技术,该技术具有快速、准确、灵敏的特点,解决了qPCR不能仅对活体病原菌进行准确鉴别和定量分析的问题,为制定有效的根肿病防控策略提供了依据。

牛呼吸疾病综合征七病原联合检测多重qPCR方法的建立

为了提高牛呼吸疾病综合征多病原混合感染的临床诊断效率,以牛支原体(Mycoplasma bovis,M.b)oppD/F基因、多杀性巴氏杆菌(Pasteurella multocida,P.m)ompH基因、溶血性曼氏杆菌(Mannheimia haemolytica,M.h)gcp基因、牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)gB基因、牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)以及牛副流感病毒(bovine parainfluenza virus type,BPIV) 3型a和c基因型(BPIV-3a,-3c)的N基因等为检测靶标,分别设计特异性引物和Taqman探针,通过优化反应条件,采用3管7联的组合方式建立了7种病原体的多联实时荧光定量PCR检测方法。结果显示,该方法仅对本试验的7种病原有特异性反应,与其他常见病原无交叉反应。对M.b、P.m、M.h、IBRV、BRSV、BPIV-3a和BPIV-3c质粒标准品的最低检测限分别为102、102、101、102、102、102和101拷贝/μL。组内变异系数小于2.5%,组间变异系数小于5.5%。平行应用该方法和常规PCR方法对临床采集的115份有呼吸道症状牛的鼻拭子进行检测,P.m阳性率36.65%,M.b阳性率27.83%,M.h阳性率25.22%,IBRV阳性率11.30%,BPIV-3c阳性率8.57%,BRSV阳性率0.95%;其中混合感染率为26.1%。共检测到11种混合感染模式,主要由M.b与其他病原体的混合感染,占72.7%(8/11);M.b/P.m混合感染的检出率最高,占60%(18/30);M.b、P.m、M.h在混合感染中出现率排前三,其占比分别为73.3%(22/30)、73.3%(22/30)和43.3%(13/30);其次为IBRV,占26.7%(8/30);BPIV-3c占13.3%(4/30)。以上结果表明,该方法具有较高的分析敏感性和特异性,可用于牛呼吸疾病综合征多病原感染的联合检测。

柑橘黄龙病常规PCR与qPCR检测体系的比较

为促进湖南省柑橘产业的可持续发展,加强对柑橘黄龙病(Huanglongbing,HLB)的监测预警,根据黄龙病菌耐热性亚洲种“Candidatus Liberibacter asiaticus”(CLas)DNA不同保守区域设计了8组引物,分别采用常规PCR和qPCR进行检测,比较2种方法的灵敏度。结果表明:常规PCR供试的4组引物中,In1/In2引物未扩增出目的条带,其他3组引物的灵敏度由高到低排列依次为OI1/OI2>LB1/LB2=OL1/OL2,OI1/OI2这组引物对HLB菌的检测下限为98 pg/μL;qPCR供试的4组引物的灵敏度由高到低排列依次为LJ900f/LJ900r>Las-I-f/Las-I-r>HLBas/HLBr>Las-O-f/Las-O-r(根据可检测到最低浓度样本的Ct值排序),其中LJ900f/LJ900r这组引物对HLB菌的检测下限为9.8 pg/μL。另外,以湖南省郴州市汝城县农业局提供的11个柑橘HLB疑似样品为材料进行检测,引物LJ900r/LJ900f进行的qPCR检测,检出率为72.7%;引物OI1/OI2进行的常规PCR,检出率为45.5%;表明引物LJ900r/LJ900f结合qPCR方法对于HLB菌的检测效率更高。

白斑综合征病毒和对虾内参基因双重荧光定量PCR检测方法的建立

【背景】白斑综合征病毒(WSSV)是一种高度传染性和致命性的病原体,对全球虾类养殖业构成重大威胁。WSSV可通过水平传播和垂直传播迅速扩散,虾在感染WSSV 3~10 d内累积死亡率最高可达到100%。由于缺乏有效的预防和治疗措施,开发快速、准确和敏感的WSSV检测技术对及时诊断和实施控制措施至关重要。【目的】建立一种WSSV和对虾内参基因双重荧光定量PCR检测方法,提高WSSV检测的灵敏度和特异性。【方法】首先,基于WSSV基因组保守序列设计合成特异性引物和TaqMan探针,建立WSSV单重荧光定量PCR检测体系,经特异性和重复性试验证实,该方法对WSSV具有良好的特异性和重复性。在此基础上,设计合成凡纳滨对虾(Litopenaeus vannamei)内参基因引物和探针,并对引物碱基进行优化替换,建立WSSV-对虾内参基因双重荧光定量PCR检测体系。【结果】敏感性试验结果显示,该双重检测体系对WSSV的检测下限可达15拷贝·μL^(-1),在6.6~6.6×10^(5)拷贝·μL^(-1)范围内,WSSV质粒标准品的起始模板量对数值与循环阈值(Ct值)呈良好的线性关系(R^(2)=0.9930)。同时,内参基因的引入可有效地区分样本中由核酸提取或操作问题引起的假阴性结果。【结论】综上所述,本研究建立的WSSV-对虾内参基因双重荧光定量PCR检测方法灵敏度高、特异性强、重复性好,可为WSSV的快速诊断及流行病学调查提供可靠的技术手段,对控制WSSV的暴发流行、保障对虾养殖业的可持续发展具有重要意义。

猫传染性腹膜炎病毒实时荧光定量PCR(冻干型)检测方法的建立

为了建立一种灵敏度高、无需冷链运输、操作简便的猫传染性腹膜炎病毒(FIPV)检测方法,本试验以FIPV的N基因序列为靶基因,进行体外转录制备阳性参考品;筛选特异性引物和探针;对反应体系和条件进行优化;筛选适宜的冻干保护剂并对本试验方法的特异性、灵敏度和重复性进行评估;对冻干体系进行稳定性试验,预测室温条件下的有效期;利用本试验方法和商品试剂盒对临床采集的11份确诊FIPV感染病料进行检测,对比二者检出率;绘制接受者操作特征(ROC)曲线,计算曲线下面积,评估本试验方法的诊断效果,计算Cut off值。结果显示,本试验方法可特异性检出FIPV;最低检测限为43 copies/mL;组内和组间重复试验变异系数分别为0.12%~1.67%和0.03%~1.62%;室温有效期至少可达567 d;本试验方法的检出率为100%,市售试剂盒的检出率为91%;ROC曲线下面积为1,Cut off值为39.40。结果表明,本试验建立的FIPV实时荧光定量PCR(冻干型)检测方法具有灵敏度高,方便运输且易于贮藏的优势,可为FIPV的临床检测和流行病学调查提供技术支持。

猫杯状病毒实时荧光定量逆转录PCR检测方法的建立

为了建立可快速检测猫杯状病毒(FCV)的方法,本试验采用肽核酸(PNA)设计分子信标荧光探针,优化反应体系和条件,建立了FCV实时荧光定量逆转录PCR(RT-qPCR)检测方法,对该方法的敏感性、特异性和重复性进行验证,并进行临床样本检测。结果显示,建立的RT-qPCR检测FCV参考株具有良好的线性关系(R^(2)=0.9995),最低检测限为1×10^(2)TCID_(50)/mL,对其他相关病毒无有效响应,组内变异系数和组间变异系数均小于1%。应用该方法检测33份临床样本,阳性率为51.5%。由此可见,本试验建立的RT-qPCR敏感性、特异性和重复性均良好,可为FCV的早期快速诊断和疫病防控等提供检测手段。

水稻细菌性条斑病菌的实时荧光PCR检测技术研究

水稻细菌性条斑病是我国水稻上的检疫性病害之一。设计并筛选出特异性引物对Xoc2071F/Xoc2071R(Xoc2071),其扩增片段大小为329 bp,利用SYBR GreenI荧光定量PCR方法进行水稻细菌性条斑菌的实时荧光PCR检测。结果表明,引物对Xoc2071能特异性检测到目标病原菌产生荧光信号而其他菌种不产生荧光信号。检测的灵敏度是2.25 fg/μL质粒DNA和1.0×102cfu/mL的菌悬液,相当于1个细菌的基因,比常规PCR电泳检测的灵敏度高约100倍。此实时荧光PCR可以检测到仅需10 g的带菌种子上目标菌的存在。