应用荧光定量PCR检测食品中沙门菌

目的应用荧光定量PCR技术检测食品中沙门菌,为病原监测和快速诊断提供实验依据。方法选择沙门菌invA基因设计引物,应用SYBRGreen I染料建立荧光定量PCR法。分别对61株沙门菌、14株非沙门菌以及食品模拟标本、市售禽蛋制品进行检测,观察方法的特异性、敏感性和可行性。结果建立的荧光定量PCR法检测所有沙门菌均出现了特异的熔解曲线,扩增产物的熔点值79.6℃左右;目标菌DNA的扩增产物循环数（Ct值）为20,空白对照及非沙门菌的Ct值大于30,非沙门菌均未出现特异的熔解曲线。每反应最低检测的沙门菌数是31 CFU;对食品中人工污染的沙门菌的检测,最低检测量是1 CFU,检测时间为7～8 h,与培养法比较,阳性符合率100%;对市售的369份禽蛋制品进行检测,阳性结果 19份,而细菌培养法检测阳性结果为12份。结论基于SYBRGreen I染料建立的荧光定量PCR检测沙门菌是敏感、特异、省时、省力的方法,适用于沙门菌快速检测。     

Taqman荧光定量PCR法在药品沙门菌快速检测中的应用

目的：建立药品中沙门菌的荧光定量PCR快速检测方法。方法：根据沙门菌的特异基因序列合成引物和探针,提取沙门菌DNA进行检测。结果：该方法特异性好,灵敏度达到160cfu·ml-1,人工污染的药品检出率为100％．结论：荧光定量PCR法可用于药品沙门菌的快速检测。     

实时荧光定量PCR法检测中药制剂中沙门菌

目的 采用实时荧光定量PCR技术,建立一种中药制剂中沙门菌的快速检测方法。方法 将中药制剂加入适量的胰酪大豆胨液体培养基中进行增菌后,取增菌液以热裂解法提取总DNA,采用实时荧光定量PCR法特异性检测沙门菌。结果 建立了中药制剂中沙门菌的实时荧光定量PCR检测法,检测可在24 h内完成,灵敏度达每10 g（10 mL）供试品1 cfu,特异性为100%,采用该方法检测18批次样品结果与药典方法一致。结论 采用实时荧光定量PCR法可明显缩短中药制剂中沙门菌检测周期,具有良好的灵敏度和特异性,可作为药典检测方法的有效补充。     

实时荧光定量PCR检测乙型肝炎血清DNA的临床观察

目的:探讨乙型肝炎患者血清乙型肝炎病毒DNA定量与乙型肝炎标志物关系.方法:采用荧光探针杂交技术为基础的实时荧光定量PCR检测259例乙型肝炎患者血清中DNA含量,与HBV标志物作对比分析.结果:血清HBeAg阳性组HBV-DNA阳性率为98.6%,HBeAb组阳性率为61.5%;HBeAg组阳性率含量明显高于HBeAb阳性组.结论:实时荧光定量PCR检测具有较高的灵敏度和特异性,定量准确、结果可靠,为临床了解病毒复制情况,制定治疗方案及疗效观察提供了确切依据.     

SYBR Green实时荧光定量PCR技术平台的建立

目的建立SYBRGreen实时荧光定量PCR技术平台。方法通过摸索SYBRGreen工作液的配制方法,经典PCR检测HBVDNA方法转为SYBRGreen实时荧光定量PCR方法及建立大鼠TGF-bate1mRNA表达水平的SYBRGreen实时荧光定量PCR方法。结果SYBRGreenⅠ工作液,40×H2O溶液稳定性好,最佳反应浓度为1∶10000。HBVDNA的SYBRGreen实时荧光定量PCR方法,最佳的MgCl2反应浓度4.0mmol,引物浓度0.5μmol,81℃时收集荧光信号。定量分析用标准曲线斜率为-3.32,相关系数为-0.994,误差为0.016。大鼠TGF-bate1表达水平的SYBRGreen实时荧光定量PCR方法,TGF-bate1和Bate-actin的最佳MgCl2反应浓度均为3.5mmol,引物浓度前者为0.8μmol,后者为0.5μmol,在87℃时收集荧光信号。靶基因TGF-bate1mRNA的相对量用下述公式计算:(1+Etgf)Ct1/(1+Eact)Ct2。结论初步建成了可检测DNA和mRNA的SYBRGreen实时荧光定量PCR技术平台。     

POLH基因实时荧光定量PCR检测条件的建立

研究表明,环境化学污染物可以引起各种类型的DNA损伤,而POLH基因与DNA损伤所引起的细胞内反应即DNA的跨损伤合成(translesion synthesis,TLS)有关.因此,有必要寻找出一种敏感度极高的方法来检测POLH基因在mRNA水平上的表达变化,从而为探讨POLH基因在环境化学污染物所诱导的跨损伤DNA合成中的分子作用机制提供相应的线索.     

多重PCR技术检测沙门菌两种四环素耐药基因

应用两对特异性引物同时检测四环素耐药基因tetB和tetC通过对35株沙门菌分离株的四环素耐药性检测，表明所有沙门菌分离株均含tetC基因，与药敏试验结果阳性符合率65．7％，8株同时含有tetB基因，与药敏试验结果阳性符合率100％，tetB基因和tetC基因双阳性的菌株与药敏试验结果阳性符合率也为100％。取其中部分菌株扩增出tetB和tetC基因片段进行序列分析，5株菌的tetB基因扩增产物序列完全相同，与质粒pRT11相应序列同源性达99．7％；14株菌的tetC基因扩增产物与质粒pBR322中的相应序列同源性为100％。证实沙门菌耐药基因普遍存在，且同时含有tetB和tetC基因的菌株表现耐药。多重PCR技术同时检测两种四环素耐药基因，适合大量样本的检测，对开展沙门菌四环素多种耐药基因的分子流行病学监测提供了有效途径。     

实时荧光定量PCR检测人乳头瘤病毒HPV 23分型的临床研究

目的：建立实时荧光定量PCR检测人乳头瘤病毒HPV 23分型的方法。方法采用实时荧光定量 PCR检测 HPV DNA。结果在60例检测患者中,有19例患者检测出HPV人乳头瘤病毒感染阳性。结论实时荧光定量PCR方法检测人乳头瘤病毒灵敏度高、特异性强,可以为宫颈癌的预防、发展监测、手术预后等提供重要的临床指导。     

细胞因子实时定量PCR检测方法的建立和应用

目的 建立细胞因子mRNA实时定量PCR的检测方法,并探讨人单个核细胞(PBMC)在激发型CD3单克隆抗体作用下,细胞因子mRNA的表达.方法 分离健康个体的PBMC,体外与激发型CD3单克隆抗体共培养,应用实时定量PCR检测不同共培养时间点Th1型(IL-2,IFN-γ)和Th2型(IL-10)细胞因子mRNA的表达.结果 激发型CD3抗体能显著上调PBMC胞浆IL-2,IFN-γ和IL-10 mRNA的表达.在共培养的12 h,IL-2 mRNA的含量显著上升并达到峰值14 000 copies/ml,随着培养时间的延长快速下降.IFN-γ和IL-10 mRNA在共培养的24～48 h达到峰值,分别为110 000 copies/ml和15 000 copies/ml.不同细胞因子其胞浆mRNA水平的基础值有明显差异.结论 实时定量PCR能在基因转录水平敏感和特异地反映微量生物活性因子的表达和调节.因此,该技术在基础和临床免疫研究中,具有良好的应用价值.     

乌梢蛇实时荧光定量PCR法的建立及与普通PCR鉴别方法的比较研究

目的 为了进一步优化乌梢蛇药材鉴别的准确性,提高鉴别速度。方法 基于CO1片段序列特征,该研究设计了乌梢蛇的特异性引物、探针,通过条件的优化建立了乌梢蛇的实时荧光定量PCR 快速鉴别方法。结果 利用该方法对正品及伪品15个物种30份样本进行了验证,结果全部8 份乌梢蛇正品均能从30 个药材样本中准确地鉴别出来。结论  该方法与药典方法比较,准确性一致,但所用设备少、检测所需要时间短,仅需40 min至1 h即可得出结果,而药典方法需要4 h左右;灵敏度高,检测限可达到1×10-3ng/反应,检测结果在0.001%~100%范围内具有良好的线性关系,相关系数R²=1,而药典方法仅为1×10-2ng/反应。该方法具有特异性强、检测速度快、灵敏度高、所需设备少的特点,适用于乌梢蛇中药材及其饮片真伪快速检测。     

实时荧光PCR法检测脑出血患者ApoE基因多态性的意义及探讨

目的探讨载脂蛋白E（ApoE）基因多态性与脑出血的关系及实时荧光聚合酶链反应（PCR）技术在ApoE基因多态性检测中的应用价值。方法对福建附一医院住院的74例脑出血患者进行研究,正常对照组为80例本院健康体检者;ApoE基因型采用实时荧光PCR方法检测,血清TC、TG、LDL、HDL在全自动生化仪上采用动力学法检测。结果脑出血患者组ApoEε4等位基因频率为14.9%,明显高于正常对照组（P〈0.01）;比较不同ApoE基因型脑出血患者的血清TC、TG、LDL、HDL含量,结果ε4组的TC含量明显高于ε3和ε2组（P〈0.05,P〈0.01）,LDL含量明显高于ε3和ε2组（P〈0.05）。结论ApoEε4等位基因可能是脑出血的易感因子和风险因素;实时荧光PCR技术检测ApoE基因多态性具有方便、快捷的优点,值得推广。     

猪源致病性沙门氏菌四环素耐药基因tetC的PCR扩增及序列分析

本研究对16株猪致病性沙门氏菌的四环素耐药性进行了检测，结果表明，沙门氏菌对四环素类抗生素产生了广泛的耐药性，耐药率达100％。对四环素耐药基因tetC进行PCR扩增，结果在12株沙门氏菌的质粒上扩增出特异性产物，与药敏试验结果阳性符合率75％，具有较高的检出率；序列分析结果表明tetC的核苷酸同源率较高，达97．7％～98．7％，建立tetC基因的PCR检测技术，为沙门氏菌对四环素耐药性的分子流行病学监测提供了有效的途径。     

Loss of imprinting of the insulin-like growth factor 2 and the H19 gene in testicular seminomas detected by real-time PCR approach

IGF2 and H19 are imprinted genes in normal human tissue, but many studies have observed a loss of imprinting (LOI) of these genes in tumors as an epigenetic alteration of the DNA, that leads to a biallelic expression predisposing cells to carcinogenesis and tumor growth. The aim of this study was to test the reliability of LightCycler™-assisted Real-time PCR in detecting LOI of IGF2 and H19 in 39 patients with testicular germ cell tumors by comparing these results with the analysis generated by the golden standard restriction fragment length polymorphism (RFLP). With LightCycler™-assisted Real-time PCR for IGF2 44% and for H19 49% of the patients were found to be heterozygous. This was consistent with the results obtained by RFLP, but surprisingly RFLP failed in more than 7% of the patients. In detecting LOI (for IGF2 in 41% and for H19 in 68% of the informative patients) the approach by RFLP was superior, since the results derived from LightCycler™-assisted Real-time PCR showed reliable results in 76 and 10% of the samples concerning IGF2 and H19, respectively. Again, no discrepancy between the results obtained by the two methods occurred. In sum, LightCycler™-assisted Real-time PCR is a sufficiently working approach for the rapid and reliable detection of heterozygosity of IGF2 or H19 gene and identification of LOI of IGF2 and thus may be helpful in conducting large epidemiological studies. However, for the identification of LOI of the H19 gene in this cohort it possesses only restrictive use.Sebastian Stier and Thomas Neuhaus contributed equally to this work.     

Detection of Ochratoxin a Using Molecular Beacons and Real-Time PCR Thermal Cycler

We developed a simple and cheap assay for quantitatively detecting ochratoxin A (OTA) in wine. A DNA aptamer available in literature was used as recognition probe in its molecular beacon form, i.e., with a fluorescence-quenching pair at the stem ends. Our aptabeacon could adopt a conformation allowing OTA binding, causing a fluorescence rise due to the increased distance between fluorophore and quencher. We used real-time PCR equipment for capturing the signal. With this assay, under optimized conditions, the entire process can be completed within 1 h. In addition, the proposed system exhibited a good selectivity for OTA against other mycotoxins (ochratoxin B and aflatoxin M1) and limited interference from aflatoxin B1 and patulin. A wide linear detection range (0.2–2000 µM) was achieved, with LOD = 13 nM, r = 0.9952, and R² = 0.9904. The aptabeacon was also applied to detect OTA in red wine spiked with the same dilution series. A linear correlation with a LOD = 19 nM, r = 0.9843, and R² = 0.9708 was observed, with recoveries in the range 63%–105%. Intra- and inter-day assays confirmed its reproducibility. The proposed biosensor, although still being finalized, might significantly facilitate the quantitative detection of OTA in wine samples, thus improving their quality control from a food safety perspective.     

Development of a quantitative PCR assay for residual mouse DNA and comparison of four sample purification methods for DNA isolation

Reliable and sensitive assays are required to determine whether a pharmaceutical product meets current regulatory guidelines for residual host cell DNA. In this study, the sensitivity of the qPCR assay was significantly improved by targeting the repetitive elements of mouse genome. This improved method allowed for sensitive and accurate quantitation of mouse genomic DNA in the range of 1 to 10⁶ pg/mL. In addition, four sample purification methods for DNA isolation (Wako DNA extractor kit, MasterPure™ DNA purification kit, PrepSEQ™ residual DNA sample preparation kit, and phenol–chloroform extraction method with addition of glycogen), each representing a different strategy for DNA isolation from proteinaceous solutions, were evaluated by isolating DNA from a mouse monoclonal IgG antibody. Among these methods, Wako DNA extractor kit and MasterPure™ DNA purification kit demonstrated superior DNA recovery, repeatability, and sensitivity, with quantitation limits of 1 pg/mL. To further evaluate these two DNA isolation methods, six replicates of an unspiked mouse polyclonal IgG antibody sample were tested by both methods, and both methods demonstrated a good degree of precision. Therefore, the residual mouse DNA quantitation methods described here represented rapid, accurate, precise, and sensitive procedures that can be used in quality control testing for regulatory compliance in the pharmaceutical industry.     

荧光定量PCR技术在基因治疗药物组织分布研究中的应用

介绍应用荧光定量PCR技术对基因治疗药物组织分布研究的实验方法。从以下几个方面,即定量PCR技术原理、实验设计思路、各种检测方法的比较来说明这一问题。其中,可以使用的检测方法包括紫外定量法和引入内参基因法,后者又可以分为外标法和内标法。充分对比了这些方法的优缺点,并结合本实验室的研究现状,介绍了本监测中心在选用外标法进行研究时所取得的一些成果和实验经验。     

Detection of HER2 Gene Polymorphism in Breast Cancer: PCR Optimization Study

Cancers are the most deadly diseases in the world and their incidences continue to increase over time. Particularly, breast cancer in females places 1^st rank among other types of cancers in term of cancer cases (23%) and death incidence (14%). Recent findings support the correlation between ^Ile655^Val SNP in the HER2 gene with breast cancer risk. Moreover, the ^Ile655^Val HER2 gene polymorphism could be a predictive factor in a neoadjuvant therapy setting. Precise detection of the ^Ile655^Val HER2 gene SNP in early breast cancer patients will be beneficial in designing the most suitable treatment and in increasing the efficacy of anticancer drugs. Here we develop a rapid and inexpensive method for ^Ile655^Val SNP detection in the HER2 gene based on allele-specific PCR technology. Two forward primers and one common reverse primer were designed to anneal specifically either on the HER2 gene fragment containing the GG genotype or to the HER2 gene fragment containing the AA genotype where one of these primers had been added with poly-GC at 5’ upstream. Moreover, to increase discrimination level, mismatch bases at the SNP site and the 3^rd base of each forward primers from 3’end were added. To test the performance of the designed primers in discriminating a polymorphism and its annealing temperature, breast cancer specimen-derived genomic DNA (with GG genotype) and pGEM_HER2/AA (with AA genotype) were used as templates in the PCR reaction. The optimal annealing temperature for SNP detection was at 51.5°C as showed by the appearance of a 150 base pair (bp) band as AA genotype (pGEM_HER2/AA template), 116bp band as GG genotype (genomic DNA template), and both types of bands as AG genotype (mix of pGEM_HER2/AA and genomic DNA template). Allelic types of breast cancer patients were also determined using this optimized method compared to sanger sequencing. The 100% accordance was shown for all types of genotypes in both methods. The allele-specific PCR in this study may have application in determining polymorphisms of the breast cancers-originated ^Ile655^Val HER2 gene.