Real-time PCR

摘要： 目的 通过研究白藜芦醇对高糖下肾小球系膜细胞hsa-miR-4689-3p、hsa-miR-4741-3p表达的影响,探讨白藜芦醇对高糖下肾小球系膜细胞的保护作用及机制。方法将肾小球系膜细胞分为空白对照组(Con),高糖组(HG)和白藜芦醇组(Res),real-time PCR技术检测hsa-miR-4689-3p、hsa-miR-4741-3p在各组的相对表达量,采用生物信息学软件预测靶基因。结果 Real-time PCR结果表明,hsa-miR-4689-3p与hsa-miR-4741-3p的相对表达量在HG组中较Con组明显降低。与HG组相比,Res组肾小球系膜细胞中的相对表达量明显升高。靶基因预测发现hsa-miR-4689有OPA3,HCN4,HPSE等75个靶基因,其中与DN密切相关的靶基因有FASN、PAX2、PTPN5、ANGPT2等,hsa-miR-4741有CILP2,MMAB,POR等53个靶基因,其中与DN密切相关的靶基因有CD4、PLVAP、GAS2L1、SMURF1、KDM4B、SERPINA3、CACNA1C、DDR1等。结论 白藜芦醇对高糖诱导肾小球系膜细胞的保护作用可能部分通过调节hsa-miR-4689-3p与hsa-miR-4741-3p实现。

摘要： 为探讨旋毛虫预感染对小鼠急性脊髓损伤(spinal cord injury,SCI)的干预效果,选取40只C57BL/6雌性小鼠随机分为4组进行实验:A组(假手术组)、B组(旋毛虫预感染+假手术组)、C组(脊髓损伤组)、D组(旋毛虫预感染+脊髓损伤组)。A组小鼠只打开椎板,不损伤脊膜及脊髓;B组小鼠以旋毛虫肌幼虫(350条/只)灌胃后14 d打开椎板;C组小鼠进行SCI造模;D组小鼠在旋毛虫肌幼虫感染14 d后进行SCI造模。在术后第7天采用Basso小鼠评分(Basso mouse scale,BMS)观察不同组别小鼠后肢运动功能变化,其后收集小鼠脊髓组织和血清,HE染色法观察脊髓损伤处病理学变化及炎性细胞浸润情况;RT-PCR分析促炎因子IL-6、TNF-α和调节性细胞因子IL-10和TGF-β基因表达水平;ELISA检测血清IL-6、TNF-α、IL-10和TGF-β分泌水平。结果显示,术后第7天,4组小鼠行为学评分及单位面积的炎性细胞浸润数量差异均有统计学意义(P<0.05);C组小鼠行为学评分明显低于A组(P<0.05),且脊髓组织结构出现明显损伤,炎性细胞浸润显著增加(P<0.05);经旋毛虫预感染干预后,D组BMS评分较C组明显改善(P<0.05),且脊髓病理损伤显著缓解,炎性细胞浸润亦明显减少(P<0.05)。RT-PCR和ELISA结果显示:与C组相比,D组小鼠脊髓匀浆和血清中促炎细胞因子IL-6和TNF-α的表达均明显降低(P<0.05);抗炎细胞因子IL-10和TGF-β的表达均明显升高(P<0.05)。结果表明,旋毛虫预感染可抑制小鼠急性SCI后的炎症反应,减轻脊髓组织病理损伤,改善SCI小鼠肢体功能。

摘要： Two real-time PCR methods for the relative quantitation of DNA from meat species in food samples are described: these methods are applicable for horse in processed beef meat products, and pork in raw/processed beef meat products. Test samples were prepared using raw meat admixtures or processed horse/pork in beef food products made to an industry-standard recipe. The methods were subjected to single laboratory method validation, evaluating the performance characteristics of specificity, PCR efficiency and r-squared (r2), Limit of Detection (LOD), Limit of Quantitation (LOQ), and precision and trueness. A limited UK-based inter-laboratory trial of the two methods was completed involving four participating laboratories. Full statistical analysis of the data qualified the applicability of the methods for accurate and sensitive trace-level analysis. The methods were deemed fit for purpose for reproducibly distinguishing between adventitious contamination at 0.1% (w/w), the level for further enforcement action at 1% (w/w), and a level representative of deliberate economically motivated adulteration (10% (w/w)). The data provided evidence that the precision of the two methods was applicable for qualitative and quantitative detection at topically important levels of adulteration. This work has added significant value to the current state of the art in quantitative determination of topical meat species adulteration, allowing analysts to distinguish between adventitious contamination and deliberate adulteration. The resulting methods described in this paper can easily be deployed and used by analytical laboratories for controls and due-diligence testing based on standard laboratory equipment.

摘要： 探讨在Hepa1-6细胞中过表达、敲低Tmem176a基因对脂质代谢途径的影响。将构建成功的Tmem176a慢病毒过表达载体、siRNA分别转染进Hepa1-6细胞中,将过表达或敲低Tmem176a后的Hepa1-6细胞进行RNA-seq、生物信息学分析、Real-Time PCR验证。与对照组相比,Tmem176a在Hepa1-6细胞中过表达182倍(P<0.05)和敲低0.2倍(P<0.05),OE-Tmem176a相较于OE-Vector差异表达基因共计424个,其中下调基因192个,上调基因共233个;KD-Tmem176a相较于KD-Scramble差异表达基因共计405个,其中下调基因248个,上调基因共157个,部分差异基因富集在脂肪的消化吸收、胆固醇代谢、甘油酯代谢、脂肪酸合成等代谢途径,Real-Time PCR验证部分差异表达的基因,差异基因表达水平的变化趋势与RNAseq一致。RNAseq结果提示Tmem176a基因会影响脂质代谢基因响应。Tmem176a表达水平改变会促进小鼠肝癌Hepa1-6细胞中脂质代谢基因表达水平发生显著变化。

摘要： 细菌性果斑病和角斑病是葫芦科作物两大重要细菌病害,病原菌分别为西瓜嗜酸菌Acidovorax citrulli和丁香假单胞菌黄瓜致病变种Pseudomonas syringae pv.lachrymans.两种菌均可通过种子、种苗带菌进行远距离传播.种子检测是预防和控制这两种病害发生的首要环节.本研究应用微滴数字PCR技术(droplet digital PCR,ddPCR)建立了同时检测种子携带西瓜嗜酸菌和丁香假单胞菌的方法.结果 显示:两种细菌菌悬液和DNA样品等浓度混合时,ddPCR能同时检测到两种靶标菌的最低混合菌悬液浓度和最低DNA浓度分别为103 cfu/mL和10-3 ng/μL,其检测灵敏度是平行测试的real-time PCR方法的10倍;对于非等浓度混合的菌悬液和DNA样品,两种靶标菌菌悬液按浓度比1∶1 000 (103∶106 cfu/mL)混合或其DNA浓度比为1∶10 000(2.28×10-3 ng/μL∶22.8 ng/μL)条件下,ddPCR可检测到低浓度的靶标菌,检测灵敏度同样是real-time PCR的10倍.此外,在人工接菌种子测试中,西瓜、甜瓜单粒种子平均带菌量105～106 cfu/粒时,ddPCR方法可检测到带菌率0.2％(n=500)的西瓜、甜瓜种子样品.将分别携带两种菌的种子按比例1∶10混合时ddPCR方法可以准确检出浓度相对低的靶标菌;而使用相同检测引物的real-time PCR检测方法则只能检出西瓜嗜酸菌和丁香假单胞菌带菌率分别为0.2％和2％(n=500)的甜瓜种子混合样品中的西瓜嗜酸菌,未能稳定检出丁香假单胞菌.综上所述,本研究基于ddPCR技术建立了可同时检测两种重要葫芦科种传细菌的方法,检测结果稳定可靠,丰富了当前种传病原细菌的检测技术体系.

摘要： 为建立粪便标本溶组织内阿米巴原虫Real-Time PCR检测方法,并评价该方法在临床粪便标本检测中的应用价值,本文基于溶组织内阿米巴原虫小亚基核糖体RNA(SSU rRNA)基因序列设计了特异性引物并使用Real-Time PCR方法对溶组织内阿米巴原虫标准株DNA进行扩增,以建立溶组织内阿米巴原虫感染的Real-Time PCR技术。同时收集临床腹泻病人粪便标本221例并分别采用镜检法、Real-Time PCR法和ELISA原虫抗原检测法进行监测,以临床诊断结果为标准,分析3种方法的特异性、敏感性等检测性能。结果表明,本文建立的Real-Time PCR溶组织内阿米巴原虫检测方法特异性好,最低检测限为0.5 fg/μL,可用于临床腹泻患者粪便标本溶组织内阿米巴原虫检测。相对而言,Real-Time PCR方法特异性和敏感性均优于镜检法和ELISA原虫抗原检测法。

摘要： 为建立粪便标本溶组织内阿米巴原虫Real-Time PCR检测方法,并评价该方法在临床粪便标本检测中的应用价值,本文基于溶组织内阿米巴原虫小亚基核糖体RNA(SSU rRNA)基因序列设计了特异性引物并使用Real-Time PCR方法对溶组织内阿米巴原虫标准株DNA进行扩增,以建立溶组织内阿米巴原虫感染的Real-Time PCR技术.同时收集临床腹泻病人粪便标本221例并分别采用镜检法、Real-Time PCR法和ELISA原虫抗原检测法进行监测,以临床诊断结果为标准,分析3种方法的特异性、敏感性等检测性能.结果表明,本文建立的Real-Time PCR溶组织内阿米巴原虫检测方法特异性好,最低检测限为0.5 fg/μL,可用于临床腹泻患者粪便标本溶组织内阿米巴原虫检测.相对而言,Real-Time PCR方法特异性和敏感性均优于镜检法和ELISA原虫抗原检测法.

摘要： 为揭示肌红蛋白基因对民猪和大白猪肉色的影响,本实验选取体重95 kg民猪、160日龄民猪和大白猪(体重95 kg,160 d)各6头,共计18头,取胸腰椎连接部背最长肌鲜肉样进行肉色检测,采用Realtime-PCR法检测民猪和大白猪背最长肌中肌红蛋白基因的表达差异.结果表明:同体重和同日龄的民猪肉色红度值均高于大白猪(P<0.05),民猪肌红蛋白mRNA表达量均高于同体重和同日龄的大白猪(P<0.05),在一定程度上说明民猪与大白猪肉色差异的主要因素是肌红蛋白含量.

摘要： 目的:本试验对肺炎支原体菌株进行定性及定量验证,以确保菌株能进行肺炎支原体相关实验.方法:肺炎支原体培养成功后,利用Real-time PCR、CCU方法鉴定肺炎支原体纯度、测定其浓度.结果:Real-time PCR结果证明此菌株为肺炎支原体,基因拷贝数达到108;CCU结果证明肺炎支原体浓度为106.结论:实验结果阐明此菌株为肺炎支原体,菌株浓度较高,本检测为肺炎支原体相关实验及其实验室诊断提供理论支持.

摘要： 为明确葡萄霜霉病菌潜伏侵染阶段的菌量与病害发生的关系,本研究对贺兰山东麓宁夏立兰酒庄酿酒葡萄园3个试验样地进行采样调查,分析3个样地检测的分子病情指数(MDI)与果园田间调查的病情指数(DI)的相关性.结果表明,宁夏立兰酒庄酿酒葡萄园3个样地的MDI和DI呈极显著相关,3个样地的MDI均与采样后15 d的DI拟合性最高;当MDI值为0.0035～0.1841时,采样后12 d葡萄霜霉病在果园零星发生.MDI大于0.0035时,可作为当地酿酒葡萄霜霉病防治预警指标.

摘要： Most of enteroviruses which are frequently found in various waters, have beenrecognized as the common causes of human infection, such as the aseptic meningitis andhand-food-and-mouth disease (HFMD) reported to be prevalent in many regions in China. Toinvestigate the occurrence of the enteroviruses in wastewater and treated wastewater, nested-PCRand the real-time PCR methods were utilized to detect the enteroviruses in three domesticwastewater treatment plants (WWTPs) in Xi’an, China. At the same time, a semi-nested PCRusing groups of specific primers was developed to examine the occurrence of pathogenic virusesof HFMD, i.e. human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), coxsackievirusA10 (CVA10). The result showed that the positive ratios for EV71, CVA16 and CVA10 were only0.06, 0.06 and 0.23, respectively, comparing with the positive ratio of 0.96 for enteroviruses. Theconcentration of enteroviruses in influent and secondary effluent trended to be stable in the rangeof 102-103copies/mL in winter, while it fluctuated in a much wider range in the spring time. Themethodology developed in this study can provide a useful tool for further investigation of theHFMD viruses’ occurrence and their relationship with enteroviruses.

摘要： Most of enteroviruses which are frequently found in various waters, have beenrecognized as the common causes of human infection, such as the aseptic meningitis andhand-food-and-mouth disease (HFMD) reported to be prevalent in many regions in China. Toinvestigate the occurrence of the enteroviruses in wastewater and treated wastewater, nested-PCRand the real-time PCR methods were utilized to detect the enteroviruses in three domesticwastewater treatment plants (WWTPs) in Xi’an, China. At the same time, a semi-nested PCRusing groups of specific primers was developed to examine the occurrence of pathogenic virusesof HFMD, i.e. human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), coxsackievirusA10 (CVA10). The result showed that the positive ratios for EV71, CVA16 and CVA10 were only0.06, 0.06 and 0.23, respectively, comparing with the positive ratio of 0.96 for enteroviruses. Theconcentration of enteroviruses in influent and secondary effluent trended to be stable in the rangeof 102-103copies/mL in winter, while it fluctuated in a much wider range in the spring time. Themethodology developed in this study can provide a useful tool for further investigation of theHFMD viruses’ occurrence and their relationship with enteroviruses.

摘要： Most of enteroviruses which are frequently found in various waters, have beenrecognized as the common causes of human infection, such as the aseptic meningitis andhand-food-and-mouth disease (HFMD) reported to be prevalent in many regions in China. Toinvestigate the occurrence of the enteroviruses in wastewater and treated wastewater, nested-PCRand the real-time PCR methods were utilized to detect the enteroviruses in three domesticwastewater treatment plants (WWTPs) in Xi’an, China. At the same time, a semi-nested PCRusing groups of specific primers was developed to examine the occurrence of pathogenic virusesof HFMD, i.e. human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), coxsackievirusA10 (CVA10). The result showed that the positive ratios for EV71, CVA16 and CVA10 were only0.06, 0.06 and 0.23, respectively, comparing with the positive ratio of 0.96 for enteroviruses. Theconcentration of enteroviruses in influent and secondary effluent trended to be stable in the rangeof 102-103copies/mL in winter, while it fluctuated in a much wider range in the spring time. Themethodology developed in this study can provide a useful tool for further investigation of theHFMD viruses’ occurrence and their relationship with enteroviruses.

摘要： Most of enteroviruses which are frequently found in various waters, have beenrecognized as the common causes of human infection, such as the aseptic meningitis andhand-food-and-mouth disease (HFMD) reported to be prevalent in many regions in China. Toinvestigate the occurrence of the enteroviruses in wastewater and treated wastewater, nested-PCRand the real-time PCR methods were utilized to detect the enteroviruses in three domesticwastewater treatment plants (WWTPs) in Xi’an, China. At the same time, a semi-nested PCRusing groups of specific primers was developed to examine the occurrence of pathogenic virusesof HFMD, i.e. human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), coxsackievirusA10 (CVA10). The result showed that the positive ratios for EV71, CVA16 and CVA10 were only0.06, 0.06 and 0.23, respectively, comparing with the positive ratio of 0.96 for enteroviruses. Theconcentration of enteroviruses in influent and secondary effluent trended to be stable in the rangeof 102-103copies/mL in winter, while it fluctuated in a much wider range in the spring time. Themethodology developed in this study can provide a useful tool for further investigation of theHFMD viruses’ occurrence and their relationship with enteroviruses.

摘要： 目的:探讨晕船适应过程中5-HT1受体mRNA的变化规律.rn 方法:大鼠模拟晕船刺激后，定位取出前庭核，应用real time PCR法检测晕船适应过程中5-HT1A、1B、1D和1F受体mRNA变化规律.rn 结果:5-HT1A、5-HT1D受体mRNA表达量在晕船刺激第1天开始升高，分别为晕船刺激前的2.32和2.71倍(P<0.01)，在晕船刺激3天后达高峰，分别为晕船前的2.54、7.63(P<0.01)，5-HT1B受体在模拟晕船刺激第1d没有显著变化，但在模拟晕船刺激第3天显著升高，为晕船前的2.72倍(P<0.01)，模拟晕船刺激7天后上述各种受体mRNA表达量均开始回落，14d接近正常水平.5-HT1F受体mRNA表达量在晕船适应前后没有显著变化。rn 结论：前庭核5-HT1A、1B和1D在晕船后1～3d一过性升高，7天后逐渐下降，14d接近正常，5-HT1受体可能在晕船的发生及适应过程中发挥重要作用.

摘要： 目的:探讨晕船适应过程中5-HT1受体mRNA的变化规律.rn 方法:大鼠模拟晕船刺激后，定位取出前庭核，应用real time PCR法检测晕船适应过程中5-HT1A、1B、1D和1F受体mRNA变化规律.rn 结果:5-HT1A、5-HT1D受体mRNA表达量在晕船刺激第1天开始升高，分别为晕船刺激前的2.32和2.71倍(P<0.01)，在晕船刺激3天后达高峰，分别为晕船前的2.54、7.63(P<0.01)，5-HT1B受体在模拟晕船刺激第1d没有显著变化，但在模拟晕船刺激第3天显著升高，为晕船前的2.72倍(P<0.01)，模拟晕船刺激7天后上述各种受体mRNA表达量均开始回落，14d接近正常水平.5-HT1F受体mRNA表达量在晕船适应前后没有显著变化。rn 结论：前庭核5-HT1A、1B和1D在晕船后1～3d一过性升高，7天后逐渐下降，14d接近正常，5-HT1受体可能在晕船的发生及适应过程中发挥重要作用.

摘要： 目的:探讨晕船适应过程中5-HT1受体mRNA的变化规律.rn 方法:大鼠模拟晕船刺激后，定位取出前庭核，应用real time PCR法检测晕船适应过程中5-HT1A、1B、1D和1F受体mRNA变化规律.rn 结果:5-HT1A、5-HT1D受体mRNA表达量在晕船刺激第1天开始升高，分别为晕船刺激前的2.32和2.71倍(P<0.01)，在晕船刺激3天后达高峰，分别为晕船前的2.54、7.63(P<0.01)，5-HT1B受体在模拟晕船刺激第1d没有显著变化，但在模拟晕船刺激第3天显著升高，为晕船前的2.72倍(P<0.01)，模拟晕船刺激7天后上述各种受体mRNA表达量均开始回落，14d接近正常水平.5-HT1F受体mRNA表达量在晕船适应前后没有显著变化。rn 结论：前庭核5-HT1A、1B和1D在晕船后1～3d一过性升高，7天后逐渐下降，14d接近正常，5-HT1受体可能在晕船的发生及适应过程中发挥重要作用.

摘要： 目的:探讨晕船适应过程中5-HT1受体mRNA的变化规律.rn 方法:大鼠模拟晕船刺激后，定位取出前庭核，应用real time PCR法检测晕船适应过程中5-HT1A、1B、1D和1F受体mRNA变化规律.rn 结果:5-HT1A、5-HT1D受体mRNA表达量在晕船刺激第1天开始升高，分别为晕船刺激前的2.32和2.71倍(P<0.01)，在晕船刺激3天后达高峰，分别为晕船前的2.54、7.63(P<0.01)，5-HT1B受体在模拟晕船刺激第1d没有显著变化，但在模拟晕船刺激第3天显著升高，为晕船前的2.72倍(P<0.01)，模拟晕船刺激7天后上述各种受体mRNA表达量均开始回落，14d接近正常水平.5-HT1F受体mRNA表达量在晕船适应前后没有显著变化。rn 结论：前庭核5-HT1A、1B和1D在晕船后1～3d一过性升高，7天后逐渐下降，14d接近正常，5-HT1受体可能在晕船的发生及适应过程中发挥重要作用.

摘要： 目的:探讨晕船适应过程中5-HT1受体mRNA的变化规律.rn 方法:大鼠模拟晕船刺激后，定位取出前庭核，应用real time PCR法检测晕船适应过程中5-HT1A、1B、1D和1F受体mRNA变化规律.rn 结果:5-HT1A、5-HT1D受体mRNA表达量在晕船刺激第1天开始升高，分别为晕船刺激前的2.32和2.71倍(P<0.01)，在晕船刺激3天后达高峰，分别为晕船前的2.54、7.63(P<0.01)，5-HT1B受体在模拟晕船刺激第1d没有显著变化，但在模拟晕船刺激第3天显著升高，为晕船前的2.72倍(P<0.01)，模拟晕船刺激7天后上述各种受体mRNA表达量均开始回落，14d接近正常水平.5-HT1F受体mRNA表达量在晕船适应前后没有显著变化。rn 结论：前庭核5-HT1A、1B和1D在晕船后1～3d一过性升高，7天后逐渐下降，14d接近正常，5-HT1受体可能在晕船的发生及适应过程中发挥重要作用.

摘要： 目的：观察通络方剂对实验性糖尿病大鼠肾组织血管紧张素Ⅰ(AngⅠ)和血管紧张素Ⅱ(AngⅡ)水平及血管紧张素Ⅱ受体1型(AT1aR)和2型(AT2R)mRNA的影响。rn 方法将3月龄健康雄性SD大鼠随机分为正常对照组(NC组，n=11)和糖尿病模型组。用链脲佐菌素(60mg/kg，一次性腹腔注射)诱导糖尿病模型。3 d后测定尾静脉末梢血糖>16.7 mmol/L为诱导糖尿病模型成功。糖尿病成模后.再将糖尿病模型组分成糖尿病通络方剂小剂量治疗组(DM+TL1组，0.5 g·kg-1·d-1，n=11)、糖尿病通络方剂大剂量治疗组(DM+TL2组，1.0 g·kg-1·d-1，n=11)和糖尿病未治疗组(DM组，n=13)。在实验第10周腹主动脉取血并取肾组织，采用放射免疫分析法测定血浆和肾组织匀浆AngⅠ、AngⅡ水平，采用Bea1—timePCB方法测定肾组织AT1aR mBNA、AT2R mRNA水平。rn 结果：糖尿病第10周时，DM组血糖明显升高，体质量明显下降，肾质量/体质量比显著增加(P值均<0.01)，DM+TL1组和DM+TL2组较DM组血糖无明显改变，部分逆转DM组体质量的减轻，均改善肾质量/体质量比的增加(P值均<0.01)。DM组血浆和肾组织匀浆AngⅠ、AngⅡ水平较NC组明显升高(P值均<0.01)，DM组肾组织AT1aR mBNA、AT2R mBNA表达较NC组显著增加(分别较NC组增加约39.0倍和4.4倍，P值均<0.01);AT1aR/AT2R比值在DM组较NC组明显升高(P<0.01)。小剂量和大剂量通络方剂干预后，DM+TL1组和DM+TL2组血浆、肾组织AngⅠ、AngⅡ水平均较DM组有不同程度降低(P值均<0.01)，且DM+TL2组较DM+TL1组血浆、肾组织AngⅠ、AngⅡ水平降低更明显(组间差异P<0.01);DM+TL1组和DM+TL2组肾组织AT1aR mBNA、AT2R mRNA表达较DM组显著降低(P值均<0.01)，且DM+TL2组较DM+TL1组肾组织AT1aR mRNA、AT2BmRNA水平降低更显著(组间差异P<0.01)。DM+TL1组和DM+TL2组肾组织AT1aR/AT2R比值较DM组不同程度降低(P值分别<0.01，<0.05)，且治疗组间无显著性差异。rn 结论：通络方剂能不同程度抑制实验性糖尿病大鼠血浆、肾组织AngⅠ、AngⅡ水平的升高，并逆转实验性糖尿病大鼠肾组织AT1aR mRNA、AT2R mBNA表达的上调和AT1aR/AT2R比值的增加。通络方剂能逆转肾质量/体质最比在实验性糖尿病大鼠的增加，但并不依赖血糖的改变。通络方剂通过对实验性糖尿病大鼠肾素-血管紧张素系统(Benin—angiotensin system，RAS)的影响可能对延缓和改善糖尿病肾病有一定的意义。

摘要： 本发明通过克隆建鲤GAPDH含内含子的部分序列，然后设计跨越内含子的引物，优化扩增反应条件，来消除DNA的污染，从而提高扩增效率。这样，可为GAPDH作为内参基因，在利用realtimePCR对建鲤功能基因的研究中提供有用的方法。

摘要： 本发明通过克隆建鲤GAPDH含内含子的部分序列，然后设计跨越内含子的引物，优化扩增反应条件，来消除DNA的污染，从而提高扩增效率。这样，可为GAPDH作为内参基因，在利用realtimePCR对建鲤功能基因的研究中提供有用的方法。