Real-time RT-PCR Assay for the detection of Tahyna Virus

A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

Real-time RT-PCR Assay for the Detection of Culex flavivirus

Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.

基因重组构建AFP mRNA的real time RT-PCR标准定量模板

目的 :构建AFPmRNA的realtimeRT PCR标准定量模板。方法 :提取肝瘤细胞株BEL740 2细胞总RNA ,进行RT PCR扩增并纯化AFP基因片段 ,与PMD 1 8T载体连接构建重组质粒。结果 :重组质粒的阳性克隆效率为81 .8% ,经酶切鉴定 ,目的基因片段已插入PMD 1 8T载体内。结论 :成功构建了AFPmRNA的标准定量模板 ,可应用于该基因的realtimeRT

Real time RT-PCR定量检测BDNF mRNA表达水平方法的建立

目的建立real time逆转录聚合酶链反应(RT-PCR)检测BDNF mRNA基因表达的方法.方法提取脑缺血组织的总RNA,进行RT-PCR扩增BDNF mRNA特异性片段,扩增产物重组到质粒上并测序,建立real time RT-PCR检测BDNF mRNA表达水平方法.结果重组的质粒经酶切和测序,目的片段已插入到载体内,得到real time RT-PCR动力学曲线.结论成功建立real time RT-PCR检测BDNF mRNA基因表达的方法.

Identification of normalization factors for quantitative realtime RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

Optimal therapeutic dose and time window of picroside II in cerebral ischemic injury

A preliminary study from our research group showed that picroside II inhibited neuronal apop- tosis in ischemic penumbra, reduced ischemic volume, and improved neurobehavioral function in rats with cerebral ischemia. The aim of the present study was to validate the neuroprotective effects of picroside II and optimize its therapeutic time window and dose in a rat model of cerebral ischemia. We found that picroside Ⅱ inhibited cell apoptosis and reduced the expression of neuron-specific enolase, a marker of neuronal damage, in rats after cerebral ischemic injury. The optimal treatment time after ischemic injury and dose were determined, respectively, as follows： （1） 2.0 hours and 10 mg/kg according to the results of toluidine blue staining; （2） 1.5 hours and 10 mg/kg according to early apoptotic ratio by flow cytometry; （3） 2.0 hours and 10 mg/kg according to immunohistochemical and western blot analysis; and （4） 1.5 hours and 10 mg/kg according to reverse transcription polymerase chain reaction. The present findings suggest that an intraperitoneal injection of 10 mg/kg picroside II 1.5-2.0 hours after cerebral ischemic injury in rats is the optimal dose and time for therapeutic benefit.

Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian immunodeficiency virus （SIV） infections. To accurately measure RNA levels of the virus, a one-step fluorescent quantitative assay was established based on the SYBR green Real-time reverse transcription-polymerase chain reaction （RT-PCR）. The lower detection limit of the assay was 10 copies per reaction for the virus. This method was successfully applied to quantify SIVmac251 and SIVmac239 viruses produced in CEMx174 cells. Additionally, the performance of the SYBR green RT-PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that the method could detect as little as 215 copies per milliliter of plasma and the dynamic pattern of viral load was highly consistent with previous results. With regard to convenience, sensitivity and accuracy our assay represents a realistic alternative to both branched-chain DNA （b-DNA） assays or real-time PCR assays based on TaqMan probes.

THE DETECTION OF MDR1 GENE EXPRESSION USING FLUOROGENIC PROBE QUANTITATIVE RT-PCR METHOD

Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cancer. Methods: The fluorogenic quantitative RT-PCR method for detection of the expression of MDR1 gene was established. K562/ADM and K562 cell lines or 45 tumor tissues from patients with lung cancer were examined on PE Applied Biosystems 7700 Sequence Detection machine. Results: the average levels of MDR1 gene expression in K562/ADM cells and K562 cells were (6.86±0.65)× 107 copies/μg RNA and (8.49±0.67)×105 copies/μg RNA, respectively. The former was 80.8 times greater than the latter. Each sample was measured 10 times and the coefficient variation (CV) was 9.5% and 7.9%, respectively. Various levels of MDR1 gene expression were detected in 12 of 45 patients with lung cancer. Conclusion: Quantitative detection of MDR1 gene expression in tumor cells was achieved by using FQ-RT-PCR. FQ-RT-PCR is an accurate, and sensitive method and easy to perform. Using this method, low levels of MDR1 gene expression could be detected in 24% of the patients with lung cancer.

相对定量real time RT-PCR检测Runx2 mRNA在甲状腺乳头状癌中的表达

目的:相对定量检测Runx2mRNA在甲状腺乳头状癌和腺瘤中的表达,并探讨其在甲状腺乳头状癌的发生发展及与钙化的意义。方法:相对定量real time RT-PCR检测14例甲状腺乳头状癌和14例甲状腺腺瘤中Runx2mRNA的表达。结果:癌组和腺瘤组的ΔCT值分别为2.395±0.302和5.028±1.179,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01);癌组和腺瘤组的2-ΔΔCT分别为7.826±5.004和1,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01)。癌组和腺瘤组内以钙化分组ΔCT结果示P>0.05,组间差异均无统计学意义。癌组按肿瘤大小<1cm和≥1cm分组,ΔCT值分别为2.629±0.300和2.212±0.124,P<0.05;2-ΔΔCT值分别为167.33±33.823和221.69±18.843,P<0.01。癌组和腺瘤组以及癌组内钙化分组TSH水平比较均P>0.05。结论:Runx2在甲状腺乳头状癌中的表达高,并与癌大小有关,在较大的癌中表达较高。Runx2与微钙化的关系,可能与甲状腺乳头状癌内钙化灶的产生及癌的发生发展有关,在其他的恶性肿瘤(如乳腺癌、前列腺癌、骨肉瘤)也有相关研究。

鲤疱疹病毒Ⅱ型对异育银鲫背鳍细胞的显微形态与免疫基因表达水平的影响

为研究鲤疱疹病毒Ⅱ型(Cy HV-2)体外感染复制特征以及异育银鲫抗病毒免疫应答反应。本实验采用组织块培养法建立了异育银鲫背鳍细胞的原代培养体系。结果显示,在10 d左右可观察到组织块迁移分离出新的单层细胞,3周左右细胞可覆盖底部面积为25cm2培养瓶的底部;经Cy HV-2悬液感染离体培养的原代细胞,3 d后病毒滴度增殖至106拷贝/m L;在病毒感染6 d后出现典型的细胞病变效应;Cy HV-2感染原代细胞后,分析前期通过鱼体水平实验鉴定出的与该病毒感染相关的免疫基因:PNP5a、MPO、MHCⅠ、LYZ-C、IL-11、ITLN、PNP5a和DUSP,Real-time Rt-PCR结果显示大部分基因在细胞水平均有显著性的上调,与鱼体水平实验结果一致。本研究建立了原代培养的异育银鲫背鳍细胞,用于构建体外感染Cy HV-2病毒的细胞模型,为深入研究Cy HV-2的感染复制规律及其与宿主的相互作用关系,以及细胞水平筛选抗病毒药物实验奠定了基础。

miR-224、miR-135a在非小细胞肺癌中的表达及与临床病理的关系

目的:检测miR-224与miR-135a两种microRNA(miRNA)在非小细胞肺癌中的表达,探讨其与肺癌临床病理的关系。方法:采用Real time RT-PCR法对31例非小细胞肺癌组织及癌旁正常肺组织的miR-224与miR-135a进行定量分析,结果由2(-△△CT)处理,并分析与临床病理资料的关系。结果:相对内参U6,miR-224在非小细胞肺癌组织中表达量为4.2761±0.8731,在正常肺组织中的表达量为0.8967±0.2154,两者相比P<0.05,miR-135a在非小细胞肺癌组织中表达量为0.3551±0.0985,在正常肺组织中的表达量为1.7443±0.3125,两者相比P<0.05。miR-224与miR-135a的表达与非小细胞肺癌的临床分期、病理分级密切相关。结论:miR-224高表达及miR-135a低表达与非小细胞肺癌的临床分期、病理分级密切相关,miR-224有可能作为非小细胞肺癌的重要肿瘤标志物。

实时荧光定量PCR检测三七SS基因表达的初步实践

运用含有SYBR Green I的Real Time RT-PCR法分析SS基因在一年生三七根、茎、芦头3个部位中转录水平的相对表达差异。统计分析表明SS基因在根中的表达量最高。本研究取得了特异性高、重复性好的结果,标准曲线斜率均在-3.33～-4范围内,扩增效率均在95%～100%之间,熔解曲线分析显示产物特异性的单一峰,为Real Time RT-PCR技术用于三七植物基因的差异表达分析建立了相应的技术平台。

TGF-β1与外阴癌变的关系

目的:研究转化生长因子-β1(TGF-β1)在外阴癌变过程中的表达及其临床意义。方法:采用realtime RT-PCR及免疫组化方法检测60例外阴鳞癌组织、60例外阴上皮内瘤样病变及10例正常外阴组织标本中TGF-β1 mRNA及其蛋白的表达情况。结果:Real time PCR检测发现,TGF-β1在外阴鳞状细胞癌中的表达量明显低于在外阴上皮内瘤样病变及正常组织中的表达量,且差异有统计学意义(P<0.05)。免疫组化结果显示,正常外阴皮肤组的TGF-β1表达阳性率高于外阴上皮内瘤样病变组及外阴鳞状细胞癌组,与两者相比有显著差异(P<0.05)。外阴上皮内瘤样病变及外阴鳞状细胞癌中TGF-β1表达的阳性率无显著差异(P>0.05)。TGF-β1的表达与外阴鳞状细胞癌患者的组织分化程度及肿瘤分期均无关(P>0.05)。结论:TGF-β1的表达可能与外阴癌变的过程有关,与外阴鳞状细胞癌的临床分期和病理类型无关。

调节糖脂代谢紊乱中药多靶点筛选的研究

[目的]观察几种中药成分对人肝癌细胞株Hep-G2糖脂代谢相关基因,包括细胞血管生成素相关生长因子(AGF)、葡萄糖-6-磷酸酶(G-6-Pase)、低密度脂蛋白受体(LDLR)和胰岛素受体底物2(IRS-2)mRNA的影响。[方法]培养人肝癌细胞株Hep-G2,以实时定量逆转录聚合酶链反应(Real Time RT-PCR)法检测中药成分对AGF、G-6-Pase、LDLR和IRS-2 mRNA的影响。[结果]小檗碱可以显著提高Hep-G2细胞AGF和LDLR mRNA的表达,姜黄素可以降低G-6-Pase mRNA的表达。[结论]小檗碱和姜黄素可能通过对糖脂代谢相关基因的调节而对糖尿病有一定的治疗作用。

KIF14 mRNA在大肠癌中的表达及其临床意义

目的探讨KIF14 mRNA在大肠癌中的表达及临床意义。方法采用实时定量聚合酶链反应技术检测104例大肠癌组织中KIF14 mRNA的表达情况(分为高表达组和低表达组),分析其与临床病理特征及预后生存的关系。结果 104例大肠癌组织中KIF14 mRNA高表达率为63.5%(66/104),RQ值为1.7~227倍,低表达率为在36.5%(38/104),RQ值为0.12~0.69。KIF14 mRNA的表达率和相对定量在不同临床分期和浸润深度、有无淋巴结转移和远处转移患者中的表达差异均有统计学意义(P<0.05)。104例大肠癌患者5年总生存率为51.9%(54/104),术后未发生转移的94例患者5年无病生存率(DFS)为44.68%(42/94)。两者生存曲线比较差异均有统计学意义(P均<0.01)。KIF14 mRNA高表达组远处转移的风险比率增加近5倍(HR 4.9;95%CI 2.8~13.8;P<0.01)。KIF14 mRNA高表达组5年总生存时间和无病生存时间均较低表达组显著降低(P<0.05)。结论 KIF14参与大肠癌的发生发展过程,可能是判断预后的新的分子生物学指标。

雌激素对荷斯坦奶牛输卵管上皮细胞中前列腺素E_2受体EP_2和EP_4 mRNA表达的影响

本实验旨在探讨奶牛输卵管上皮细胞中是否存在前列腺素E2受体EP2和EP4,且该受体mRNA表达是否受雌激素(E2)的调控。将前列腺素类化合物PGE2和受体选择性激动剂(butaprost)按10-9mol/L-10-5mol/L的浓度分别作用于体外培养的奶牛输卵管上皮细胞,应用Elisa方法检测细胞中第二信使cAMP量的变化。然后,将E2作用于体外培养的奶牛输卵管上皮细胞,应用real time RT-PCR技术检测EP2和EP4受体mRNA表达量的变化。Elisa实验结果显示,前列腺素类化合物PGE2和受体选择性激动剂(butaprost)可引起奶牛输卵管上皮中cAMP量的变化,且cAMP量具有对PGE2和butaprost浓度依存性的变化规律,表明奶牛输卵管上皮细胞中存在前列腺素受体EP2和EP4。Real time RT-PCR实验结果表明,奶牛输卵管上皮细胞中存在前列腺素受体EP2和EP4,且E2对EP2和EP4受体mRNA的表达具有调控作用,低浓度(10-12mol/L)可提高该受体mRNA的表达量。

载脂蛋白J在实验性脑缺血再灌注大鼠脑内的表达

目的探索载脂蛋白J(Apo-J)及其mRNA在大鼠脑缺血再灌注模型各脑区的表达情况及其可能的作用机制。方法清洁级Wistar成年大鼠150只,首先分为空白对照组(NC组,10只)、假手术组(SO组,70只)、缺血再灌注组(IR组,70只);后两组再随机分为造模后3h、6h、12h、1d、3d、5d、7d各7个亚组,每亚组10只。NC组及以上各亚组再各自随机分为RT-PCR组(5只)、免疫组化及TUNEL组(5只)。制作脑缺血再灌注(IR)模型,用Real Time RT-PCR检测IR区及其前后脑区Apo-J mRNA的表达;用免疫组化检测IR组再灌注周边区及SO组、NC组与之对应的相同区域Apo-J的表达,并用TUNEL法检测前述免疫组化检测区的细胞凋亡情况。结果Apo-J mRNA在IR周边区表达增加,各时点中以IR 1d亚组最高(P<0.01),各亚组均显著高于相同时点的SO组、NC组(P<0.01);并且与再灌注前区、后区比较,均显著增加(P<0.01)。Apo-J在IR组再灌注周边区的表达增加,以1d亚组最高(P<0.01),各亚组均显著高于相同时点的SO组、NC组(P<0.01)。TUNEL显示了与Apo-J表达平行的细胞凋亡情况。结论大鼠脑缺血再灌注后再灌注局部Apo-J mRNA、Apo-J的表达增加,且其增加与细胞凋亡增多有关,推测局部表达的Apo-J可能起保护细胞、延缓细胞凋亡的作用。

HMGB1在非小细胞肺癌中的表达及与临床病理的关系

目的检测高迁移率族蛋白B1(HMGB1)在非小细胞肺癌(NSCLC)组织中的表达,探讨其与临床病理学特征的关系。方法采用Real time RT-PCR法及免疫组织化学染色法检测64例NSCLC组织及20例癌旁正常肺组织中HMGB1mRNA和蛋白表达,并分析与临床病理资料的关系。结果 NSCLC组织中HMGB1阳性率显著高于正常肺组织,HMGB1的平均光密度值和HMGB1mRNA的相对转录水平均显著高于正常肺组织,差异均有统计学意义(P<0.01)。HMGB1在鳞癌组织中的平均光密度值显著高于腺癌组织,在有淋巴结转移NSCLC组织中的平均光密度值显著高于无淋巴结转移NSCLC组织,差异均有统计学意义(P<0.01和P<0.05)。HMGB1在组织学低分化组表达高于高分化组,随着临床分期增高HMGB1表达升高。HMGB1在NSCLC组织中的表达与患者年龄、性别无关。结论 HMGB1高表达可能与NSCLC的发生、发展及预后不良有关,检测HMGB1表达水平对NSCLC早期诊断及预后的综合评价有一定指导意义。

采用PCR技术检测植物病原菌

聚合酶联反应(PolymeraseChainReaction,PCR)用于各类DNA、RNA的体外扩增,具有灵敏、特异、快速等诸多优点,目前已广泛地作为人类、动植物病毒、真菌等分子检测的重要手段.并在此基础上与分子生物学其他最新技术相结合,进一步发展了IC-PCR,realtimefluorescentPCR,PCR-ELISA等复合PCR技术.将PCR技术与这些方法的突出优点相结合使用,从而提供了更快捷、特异、准确、高效检测侵染植物病原菌的方法.

人小细胞肺癌细胞H446 Snail基因表达及其在顺铂耐药中的作用

目的观察锌指转录因子snail在耐顺铂(cisplatin,CDDP)的人小细胞肺癌(small cell lung cancer,SCLC)H446/CDDP细胞中的表达情况,探讨snail表达在人SCLC顺铂耐药中的作用。方法培养H446细胞系和H446/CDDP细胞系,采用MTT法检测H446/CDDP的耐药指数;分别提取人SCLC顺铂耐药细胞H446/CDDP及其亲代细胞H446的mRNA及总蛋白,应用sybgreen实时荧光RT-PCR、Western blot方法检测snail mRNA及蛋白表达水平。结果 H446/CDDP细胞snail蛋白及mRNA表达水平较H446细胞显著性升高(P<0.05)。结论 SCLC的顺铂耐药性可能与snail表达水平增高有关。