Real Time PCR研究进展及其在海洋病原生物检测中的应用

Real Time PCR,即实时监测PCR扩增产物并进行解析的方法,目前已广泛应用于分子生物学研究的各个领域。Real Time PCR技术秉承及发展了普通PCR的快速、高灵敏度检出等优点,同时克服了普通PCR不能准确定量、容易污染等缺点,无需在反应结束后通过电泳操作确认扩增产物。目前,Real Time PCR可设计多对引物在同一反应体系中同时对多个靶基因进行扩增,实现多重实时定量检测。Real Time PCR使PCR技术发生了质的飞跃,扩展了PCR技术的应用范畴,是一种具有划时代意义的技术。本文主要介绍Real Time PCR的主要原理、解析方法、技术发展趋势及其在海洋病原生物检测方面的应用。

转基因番茄植株Mi1.2表达水平的Real Time PCR检测体系的建立

[目的]为转基因番茄植株的高通量筛选奠定基础。[方法]利用CTAB法提取番茄叶片总RNA进行Real Time PCR扩增，分析转MI1、2基因的番茄植株表达水平的检测体系。[结果]提取RNA的A260／A280为1．78～1．88，RNA无明显降解。在严谨扩增条件下，引物SYBR2的扩增效率高于SYBR1。Mg^2＋的适宜浓度为2．0mg／L。Real Time PCR扩增产物具有良好的特异性，熔解曲线特异峰出现在84．5℃附近，在熔解曲线略低于83℃附近有极微弱的非特异峰。因此在定量反应中信号检测步骤应放在84℃。以4种不同模板分子数条件下扩增曲线Ct值得到的回归方程为Y=-3．78×log（copynumber）＋39．50，相关系数为0．998。[结论]该试验获得的Real Time PCR体系可用于转基因植株表达水平的检测。

应用Real Time PCR方法检测病毒灭活去除效率

目的建立检测血液制品中病毒灭活去除效率的模型。方法应用SYBR GREEN I实时荧光定量PCR验证不同方法的病毒去除效率,并用病毒感染力实验验证不同方法的病毒灭活效率。结果以PCR产物为外参的实时荧光定量PCR正确反映出实际核酸含量和检出拷贝数的线性关系,以重组质粒为外参的实时荧光定量PCR实验证明巴氏灭毒法的病毒去除效率低于金磁颗粒法。病毒感染力实验证明巴氏灭毒法灭活效率略高于金磁颗粒法。结论实时荧光定量PCR法联合病毒感染力试验可以有效评价不同方法在病毒灭活和病毒去除方面的差异。

转基因草莓植株cNHX1基因表达水平和拷贝数的Real Time PCR分析

分析了转cNHX1基因的草莓不同植株的外源基因表达水平和cNHX1基因的拷贝数。结果表明:不同转基因株系cNHX1基因表达水平存在差异,而且这种差异随盐胁迫时间的延长而更明显。在6个株系中,T6株系有最高和最稳定的cNHX1基因表达水平,而T2株系的表达水平最低。对转基因植株基因组cNHX1基因拷贝数的分析表明,T3、T6、T8和T14的含量相似,而与T2株系相差289%～310%,推测T3、T6、T8和T14株系为单拷贝植株,而T2株系为3拷贝株系。

微小隐孢子虫和安氏隐孢子虫SYBR Green real time PCR检测方法的建立

根据GenBank中登录的隐孢子虫小亚基rRNA基因序列设计1对引物,并以标准基因组DNA为模板,初步建立了检测乳牛微小隐孢子虫和安氏隐孢子虫的SYBR Green real time PCR方法,并对乳牛微小隐孢子虫和安氏隐孢子虫阳性样品和上海40份乳牛粪便进行了检测。结果表明,此次建立的real timePCR对微小隐孢子虫和安氏隐孢子虫均能扩增出曲线,且其他寄生虫(鸡贝氏隐孢子虫、刚地弓形虫、犬新孢子虫)和大肠杆菌均未检测到;标准基因组DNA的检测阈值达到5个拷贝,牛粪中卵囊的最低检测量为每克粪便5个卵囊,乳牛粪便阳性率为15%(6/40)。表明,建立的荧光定量PCR快速、特异、敏感,可用于乳牛隐孢子虫病的流行病学调查。

Real Time PCR监测慢性粒细胞白血病bcr/abl融合基因的临床意义

目的通过Real Time PCR实时监测慢性粒细胞白血病bcr/abl融合基因的表达,探讨其在诊断及预后评价价值。方法采用荧光定量Real Time PCR反应技术检测39例慢性粒细胞白血病及其它血液系统疾病20例的bcr/abl融合基因表达水平。结果骨髓原始、外周血原始与bcr/abl融合基因拷贝数有显著相关性,初诊慢性粒细胞的bcr/abl融合基因表达水平普遍明显增高,经羟基脲±干扰素治疗后表达明显下降,复发或加速期患者可提前2-4个月左右早期发现,格列卫或移植治疗后,多数bcr/abl融合基因可转阴性。结论 bcr/abl融合基因检测对诊断慢性粒细胞白血病有重要价值,同样是慢性粒细胞白血病治疗和预后观察的决定性指标。

Real time PCR检测L1-CAM在大肠癌中的表达及其临床意义

目的探讨L1细胞黏附分子(L1-CAM)在大肠癌中的表达情况及其与临床病理的相关性。方法应用实时荧光定量PCR检测浙江省人民医院58例大肠癌组织及其癌旁正常组织中L1-CAM的表达,分析L1-CAM在大肠癌中的表达及其与临床病理学特征和预后的关系。结果癌组织和癌旁正常组织L1-CAM m RNA表达差异有统计学意义(P<0.05)。癌组织L1-CAM m RNA表达与肿瘤分化程度、静脉侵犯、淋巴管侵犯、淋巴结转移、Dukes分期密切相关(均P<0.05),而与性别、年龄、肿瘤大小、肿瘤类型无明显相关(均P>0.05)。结论 L1-CAM在大肠癌组织中高表达,并且与大肠癌的浸润、转移有关。

Real time PCR与常规血培养在血流感染未知病原体鉴定中的比较研究

目的观察real time PCR在血流感染病原体检测中的敏感性和特异性,并与常规血培养对比,探讨其临床应用价值。方法以该院各临床科室收集的108份脓毒血症患者血液标本进行real time PCR检测,同时进行常规血培养,比较两种方法的特异性和敏感性。结果 108份标本当中,两种方法检测出12种病原微生物。Real time PCR共检测出阳性标本25份,阴性标本83份。其中与血培养共同阳性标本9份,共同阴性标本78份。两方法的一致性为80.6%。Real time PCR的阴性预测值是0.94,敏感性64%,特异性83%。16例标本real time PCR阳性而血培养阴性,5例标本血培养阳性而real time PCR阴性。同时,有2病标超出real time PCR的检测范围,而血培养阳性。此外,real time PCR无法检测光滑念珠菌。结论 real time PCR虽然能快速检测血液感染中病原微生物,但依然不能完全替代血培养。

Diagnosis of Toxoplasma gondii infection in pregnant women using automated chemiluminescence and quantitative real time PCR

Objective: To identify serodiagnosis and quantification of Toxoplasma gondii(T. gondii) infection among pregnant women in Salmas, northwest of Iran. Methods: In this crosssectional study, 276 blood samples were collected from pregnant women referred to the health care centers in Salmas city. The demographic variables were also recorded. Titers of antiToxoplasma IgM and IgG antibodies(Ab) were determined using the chemiluminescence immunoassay. Quantitative real-time PCR targeting the T. gondii repeated element gene was also performed on the blood sample. Results: Out of all, 19.92%(55/276) and 2.17%(6/276) patients were seropositive for anti-Toxoplasma IgG and IgM Ab, respectively. Moreover, the presence of T. gondii DNA was observed in 12.31%(34/276) blood samples. A significant relationship was observed between the IgG Ab seropositivity and contact with the cat as a risk factor(P=0.022). Conclusions: The seroprevalence rate of T. gondii infection in pregnant women is relatively low. Consequently, the seronegative pregnant women are at risk, and a considerable rate of positive blood samples for the presence of parasite's DNA should not be ignored. Besides, quantitative real-time PCR could be considered as an accurate method for diagnosis of acute toxoplasmosis especially when the precise results are of the most importance in pregnancy. Limiting contact with cats is also suggested for pregnant women.

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis

Objective This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay(MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria(diarrhoeagenic Escherichia coli, Salmonella, and Shigella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2). Methods A two-tube MCMRT-PCR assay was performed on 7900 HT Fast Real-Time PCR System(Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. Results The detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mL for S. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL for Salmonella, 2.5×102 CFU/mL for Shigella, 2.1×102 CFU/mL for V. parahaemolyticus, and 1.2×102 CFU/mL for E. coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL. Conclusion A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention(CDC).

A Duo 4-Plex Real Time PCR for Detection of Eight Tick-Borne Zoonoses in Kenya

Ticks harbor multiple pathogens, most of which can be transmitted to humans. The ensuing zoonoses display non-specific symptoms that make definitive diagnosis difficult. We report here the development and evaluation of multiplex real time polymerase chain reaction (qPCR) assays for eight tick-borne zoonoses (TBZ). The assays were organized in duo formats of 4-plex each. Format 1 was optimized for Anaplasma phagocytophilum, Coxiella burnetii, Borrelia burgdoferi and Ehrlichia chaffeensis. Format 2 was optimized for Rickettsia species (spp.), Bartonella spp., Borrelia spp. other than B. burgdoferi and Babesia spp. Synthetic plasmids were used to show that the assays can specifically detect all target sequences in the same reaction tube. Assays were assayed eight times to determine assay performance and the limit of detection was determined as the lowest plasmid concentration that was amplified for all the targets. Standard curves of threshold cycle (Ct) versus copy numbers were generated and used to determine linearity and efficiency of the assays. Pairwise comparison of singleplex and multiplex assays was done using Bland-Altman plots. Prevalence was calculated as overall percentage of positive patients to each TBZ tested Assay 1 had a limit of detection of 2 copy numbers for all targets. Assay 2 was less sensitive and on average had a limit of detection of 18 gene copies. In replicate tests, both assays had intra-assay variation of less than two cycles. Multiplex assays performance was comparable to respective singleplex assays. Evaluation of 512 clinical samples collected between 2008 and 2016 from acute febrile illness patients attending hospitals in different counties in Kenya revealed a 20% prevalence of tick-borne pathogens comprising B. burgdorferi (6%), non B. burgdorferi Borrelia spp. (3%), C. burnetii (5%), A. phagocytophilum (5%), Rickettsia spp. (2%), E. chaffeensis (0.8%), Bartonella spp. (0.8%), and Babesia spp. (0.4%). The high analytical sensitivity suggests potential for the duo 4-plex qPCR for detection of common TBZ.

Selection of Reference Genes in Equine White Blood Cells for Real Time PCR Normalization Following Extracorporeal Shock Wave Therapy

Selection of proper reference genes (RGs) is an essential step needed for accurate normalization of results from genomic studies. Expression of RGs is regulated by many factors such as species, age, gender, type of tissue, the presence of disease, and the administration of therapeutic treatment. The aim of the present study was to identify optimal RGs in a set of blood samples collected at different time points (0, 24, 48, 72 h) from horses following administration of extracorporeal shock wave therapy (ESWT). The mRNA expression of twelve RGs: HPRT1, ACTB, HSP90A, SDHA, GUSB, B2M, UBC, NONO, TBP, H6PD, RPL32, GAPDH was determined using real time quantitative polymerase chain reaction (qPCR). An SAS program developed on the algorithm of geNorm, SASqPCR, was used to determine stability of the expression and the number of optimal RGs. The results showed that the range of quantification cycle (Cq) values of the evaluated genes varied between 17 and 26 cycles, and that one optimal RG, ACTB, was sufficient for normalization of gene expression. Results of stability of expression demonstrated that ACTB was the optimal choice for all the samples studied. Notably, in samples collected at 72 h post ESWT, TBP showed a significant change in the expression level, and was not suitable for use as a RG. These results substantiate the importance of validating and selecting an appropriate RG.

Quantitative Detection of <i>Helicobacter pylori</i>by Real Time PCR in Drinking Water—Environmental and Public Health Risk Significance

Helicobacter pylori (H. pylori) is bacteria considered to be present in half of the population and it is a public health problem worldwide. Most patients infected with H. pylori show no clinical symptoms;nonetheless, approximately 10% to 20% of these patients will develop peptic ulcers and 1% will develop gastric cancer. The International Agency for Research on Cancer has classified H. pylori as a Group 1 carcinogen, recognized as the only bacteria capable of producing cancer. Samples of drinking water (n = 44) from aqueducts with chlorination treatment in selected areas with high prevalence of gastric cancer were analyzed in Costa Rica. Samples of drinking water from Panamá (n = 44) from aqueducts supplying untreated water for human consumption in the province of Chiriquí were also analyzed. The molecular marker of H. pylori, glmM, was used, and to optimize the Real Time PCR (qPCR) technique, annealing temperature, concentration of primers and probe were standardized;also, by analyzing different standard curves, the best reaction conditions that allowed detecting and quantifying the gene were determined. The LightCycler&reg 480 II (LC480II) equipment from Roche Diagnostics GmbH was used, as well as the Absolute Quantification Analysis by means of the Second Derivative Maximum Method. In the case of the samples from Costa Rica, it was determined that 79.5% were positive for H. pylori;removing outlier high average, quantification of bacteria was determined in 3.6 × 103 copies/100 mL. For Panamá it was determined that 86% of the samples were found positive for the presence of H. pylori;removing outlier high average quantification of bacteria was determined at 3.3 × 102 copies/100 mL. The difference in values between the aqueducts in both countries revealed an environmental distribution of the bacteria of epidemiological interest in each case.

Rapid Identification of Methicillin Resistant <i>Staphylococcus aureus</i>Using Real Time PCR

Screening for colonization with methicillin resistant Staphylococcus aureas (MRSA) is a key aspect of infection control to limit the nosocomial spread of this organism. Current methods for the detection of MRSA in clinical microbiology laboratories using conventional methods is time consuming. In this research we are trying to evaluate the use of real time PCR for the detection of MRSA. The PCR assay was evaluated in clinical isolates of MRSA (n = 45) and methicillin susceptible Staphylococcus aureas MSSA (n = 10). The diagnostic values of the assay showed high sensitivity and specificity. This real-time PCR assay proved to be a fast, sensitive and specific tool for MRSA detection in a routine microbiological laboratory. Real-time PCR now is available in all laboratories so its use in identification of MRSA will help in shortening the period for MRSA identification and will help in the success of infection control programs in hospitals.

基于病菌孢子捕捉和real-time PCR技术的田间空气中小麦白粉病菌孢子动态监测及病情估计模型研究

利用Burkard定容式孢子捕捉器结合real-time PCR定量技术,分别对种植高抗、中感和高感白粉病小麦品种的田间空气中白粉病菌分生孢子浓度进行监测,结果表明,real-time PCR定量与传统的显微观察计数两种方法测得的孢子浓度呈显著正相关(P≤0.01),且两种病菌孢子计数方法在同一抗性品种上监测到的孢子浓度动态相近。此外,两种方法测得的孢子浓度与各气象因子的相关性分析结果一致,空气中的白粉病菌孢子浓度主要与空气相对湿度显著正相关。在此基础上,利用两种方法测定的田间空气中白粉病菌孢子浓度分别建立了基于累积孢子浓度的田间病情估计模型。分析发现,基于两种孢子浓度测定方法建立的病情估计模型间无显著性差异,表明real-time PCR定量技术测定的孢子浓度在构建白粉病病情估计模型上具有一定可行性。该结果为real-time PCR定量技术与病菌孢子捕捉技术相结合用于小麦白粉病的监测和预测提供理论依据。

基于Real-time PCR法检测乳粉中牛源性成分定量研究

基于Real-timePCR建立了乳粉中牛源性成分相对定量检测方法,并对牛的特异性引物与探针进行了特异性、灵敏度和稳定性测试。通过模拟不同浓度牛乳粉与马乳粉混合样本,根据其△Ct值的函数关系进行线性拟合进而绘制标准曲线,建立乳粉中牛源性成分的相对定量检测。结果显示,该方法的最低检测限为0.00001 mg/mL,回收率为91.11%~119.2%,组间变异系数≤0.58%、组内变异系数≤1.44%。说明该方法在特异性与稳定性上适用于乳粉中牛源性成分及含量的掺假检测。

一种基于real-time PCR技术的TTV检测方法的建立及应用

目的:本研究旨在开发一种具有更高灵敏度和特异性的TTV检测技术,为揭示TTV在多种疾病过程中的作用提供重要的技术支持。方法:为了更精确、灵敏的检测TTV,本研究分析了目前公布的所有亚型的TTV基因序列,在此基础上建立了一种基于UTR区域的real-time PCR检测方法,并与文献报道应用较为广泛的PCR检测方法进行了对比。结果:本研究建立的方法在1×10^(7)~1×10^(1) copies/μL标准品浓度范围内具有良好的线性关系,相关系数为1.000,斜率为-3.446,检测下限为1×10^(1) copies/μL。重复性试验结果显示,组内变异系数为7.22%,表明本方法重复性、稳定性较强。针对30份临床样本,使用本研究建立的real-time PCR检测方法及目前被多个研究所使用的4套引物进行对比。结果表明,本研究所建立的方法灵敏度显著高于文献中报道的4种方法(P<0.01);Sanger测序结果表明,本方法检测出的30份阳性样本均为TTV,检测特异性为100%。结论:本研究采用基于TaqMan探针的real-time PCR检测方法,检测灵敏性高、覆盖基因型范围广,尤其对于TTV病毒载量较低的情况下能够进行定量检测,对于TTV病毒的致病性及作为免疫标志物的应用提供重要的技术支持。

24重荧光real-time PCR技术在食物中毒快速检测中的应用

目的:应用24重荧光real-time PCR检测技术快速筛检食物中毒病原菌,结合国家标准中的培养法探讨24重荧光real-time PCR检测技术符合性和应用价值。方法:采用高灵敏度、高特异性的24重荧光real-time PCR检测技术作为中毒病原菌的初筛方法,国标方法进行细菌分离培养,并对分离出的病原菌进行生化鉴定。结果:5份食物中毒患者肛拭子在增菌前检出4份霍乱弧菌核酸(非O1/非O139群,24重荧光real-time PCR),9份患者肛拭子在增菌后检出5株霍乱弧菌(非O1/非O139群,国标培养法),其中包含4份PCR技术初筛阳性样品,两个方法的符合率为80%。所有样本均未检出沙门氏菌、志贺菌、副溶血性弧菌、致泻性大肠埃希菌以及金黄色葡萄球菌。结论:应用24重荧光real-time PCR检测技术同时检测24种常见致病病原菌,能高效锁定中毒病原菌。将其与国标培养法相结合,对临床治疗和食物中毒快速处置能起到积极作用,值得应用和推广。

肉中猪源性成分Real-time PCR定量检测技术

【目的】建立一种快速、准确的肉中猪源性成分定量检测方法。【方法】首先从GenBank数据库中筛选猪特异性的微卫星DNA,根据微卫星DNA核酸序列设计引物,对常见10种动物基因组DNA进行PCR扩增,通过有无扩增产物判断筛选的微卫星DNA对猪源性成分的特异性。然后根据微卫星DNA核酸序列,设计特异性引物和探针,建立猪源性成分Real-time PCR检测方法,采用双标准曲线分别对猪源性成分和总动物源性成分进行定量,计算猪源性成分的百分含量。【结果】筛选到猪特异性微卫星DNA(Accession EF172428),根据其序列设计的引物SEQ-sus2-F/R只能从猪基因组DNA中扩增出目的条带,其他动物的基因组均无目的条带扩增。建立的Real-time PCR检测方法灵敏度为0.02 ng/25μL反应体系。该方法能够准确检测出混合DNA样品中猪源性成分和混合肉样品中猪源性成分,百分误差分别约为1.32%和1.06%-7.12%。【结论】本研究利用Real-time PCR技术建立的定量猪源性成分的检测方法可以用来检测猪源性成分在混合样品中的百分含量。

猪NLRP3基因Real-time PCR检测方法的建立及应用

根据NCBI NLRP3 cds(NM-001256770)序列设计1对特异性引物,构建标准品质粒pMD18T-NLRP3-189。通过反应条件优化、稳定性、敏感性等试验,成功建立了NLRP3的SYBR GreenⅠreal-time PCR检测方法,用该方法定量检测猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)感染后的猪肺组织中NLRP3表达水平。最佳引物浓度为0.15μmol/L,建立的标准曲线方程为y=-3.5196x+36.968,E值为0.96,最低检测量为84个拷贝,熔解曲线有单一峰,批内变异系数CV为0.067%~0.184%,批间变异系数为0.221%~0.594%,重复性好。该方法检测仔猪在感染Mhp后肺组织中的NLRP3 mRNA表达水平,感染组与健康组相比差异显著(P<0.01),NLRP3表达水平明显增高,在感染后14 d可达到高峰,平均mRNA拷贝数达18000左右,可持续42 d。表明该检测方法稳定性良好,精确度高,特异性强,为进一步研究猪炎症小体NLRP3的激活及其相关炎症反应机制提供了技术支持。