SYBR GreenⅠ荧光RT-PCR检测犬瘟热方法的建立和应用

根据GenBank发表的犬瘟热病毒(CDV)的F基因序列,经过分析在F片段的保守区域内设计引物,建立了SYBR GreenⅠ荧光RT-PCR检测CDV的方法,并通过对厦门市宠物医院收集的临床发病和疑似发病的犬病料(包括眼分泌物、鼻拭子、唾液、血液、尿液等)的检测,结果表明,本研究建立的快速检测CDV的SYBR GreenⅠ荧光RT-PCR方法具有特异性强、灵敏度高、操作简便等优点,值得推广应用.     

基于SYBR Green I的双链DNA定量方法

摘要 基于SYBR Green I荧光染料与双链DNA（dsDNA）结合产生荧光的原理,建立一种高精度、高通量的双链DNA 定量方法。将梯度稀释后的基因组DNA及已知浓度的?DNA与等体积的SYBR Green I（4×）充分混合后,利用荧光定量PCR仪采集荧光信号,以ROX（1×）作为校正染料进行定量分析;同时利用紫外分光光度计对样品进行平行测定,比较该方法与紫外分光光度法的检测限与准确度。紫外分光光度法的检测限为2 ng/?l,而SYBR Green I荧光定量法的检测限可达到0.015 ng/?l,并且在0.015~2 ng/?l范围内,SYBR Green I荧光强度与?DNA浓度呈线性关系（R2=0.9999）,比紫外分光光度法灵敏100倍以上,并可准确定量低纯度的DNA样品。此方法具有重复性好、高通量的特点,仅需少量的生物样本即可满足定量要求,为分子生物学研究及临床检验等多个领域提供了一种可靠的dsDNA定量方法。     

SYBR Green I实时荧光定量RT-PCR测定猴免疫缺陷病毒RNA拷贝数方法的建立

目的建立SYBR Green I荧光染料实时定量RT-PCR方法,测定猴免疫缺陷病毒(SIV)RNA拷贝数。方法巢式RT-PCR扩增SIV病毒RNAgag基因上1360-1837之间的长度为477 bp的片段,将该片段克隆到pGEMT载体上,构建pGEM-SIVgag477质粒。该质粒经限制性内切酶NotⅠ酶切后,进行体外转录,转录出的RNA产物(RS)纯化后10倍系列稀释,作出标准曲线,作为SIV病毒RNA荧光定量检测的外标准品。结果应用Qiagen公司QuantiTect SYBR GREEN RT-PCR Kit,该标准品可精确定量到100 copies/μL。结论制备的RS外标准品纯度高,SYBR Green I荧光染料实时定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒(SIV)RNA拷贝数。     

SYBR Green Ⅰ实时荧光定量RT-PCR测定猴免疫缺陷病毒RNA拷贝数方法的建立

目的 建立SYBR Green Ⅰ荧光染料实时定量RT-PCR方法,测定猴免疫缺陷病毒(SIV)RNA拷贝数.方法 巢式RT-PCR扩增SIV病毒RNA gag基因上1360-1837之间的长度为477 bp的片段,将该片段克隆到pGEM T载体上,构建pGEM-SIVgag477质粒.该质粒经限制性内切酶Not I酶切后,进行体外转录,转录出的RNA产物(RS)纯化后10倍系列稀释,作出标准曲线,作为SIV病毒RNA荧光定量检测的外标准品.结果 应用Qiagen公司QuantiTect SYBR GREEN RT-PCR Kit,该标准品可精确定量到100 copies/μL.结论 制备的RS外标准品纯度高,SYBR Green Ⅰ荧光染料实时定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒(SIV)RNA拷贝数.     

SYBR Green Ⅰ荧光RT-PCR法检测贝类中的诺如病毒

针对诺如病毒II型的保守区域设计引物,建立了SYBR Green I实时荧光RT-PCR检测诺如病毒II型的反应体系。此方法的病毒检测下限达到102拷贝,标准曲线的线形范围为102~106拷贝,相关系数为0.9952,斜率为?2.982,截距为35.84。对诺如病毒II型检测特异,与轮状病毒、腺病毒、甲肝病毒、星状病毒无交叉反应。针对质粒标准品检测的批内试验变异系数(CV)为0.95%~1.69%(n=5),批间试验CV为0.87%~1.24%(n=3)。运用此方法随机检测30份贝类水产品,检测出2份阳性样品。结果表明,SYBR Green I荧光RT-PCR检测诺如病毒II型的方法灵敏、特异、重复性好,可应用于贝类水产品的快速检测。     

SYBR Green I荧光RT-PCR法检测贝类中的诺如病毒

针对诺如病毒II型的保守区域设计引物,建立了SYBR Green I实时荧光RT-PCR检测诺如病毒II型的反应体系。此方法的病毒检测下限达到102拷贝,标准曲线的线形范围为102~106拷贝,相关系数为0.9952,斜率为?2.982,截距为35.84。对诺如病毒II型检测特异,与轮状病毒、腺病毒、甲肝病毒、星状病毒无交叉反应。针对质粒标准品检测的批内试验变异系数 (CV) 为0.95％~1.69％ (n=5),批间试验CV为0.87％~1.24％ (n=3)。运用此方法随机检测30份贝类水产品,检测出2份阳性样品。结果表明,SYBR Green I荧光RT-PCR检测诺如病毒II型的方法灵敏、特异、重复性好,可应用于贝类水产品的快速检测。     

SYBR GreenⅠ实时荧光定量RT-PCR测定肠道病毒71型（EV71）RNA拷贝数方法的建立

目的建立SYBR GreenⅠ荧光染料实时定量RT-PCR方法,测定实验动物等来源的EV71病毒RNA。方法运用EV71VP1保守区引物,优化real time RT-PCR条件,运用NASBA方法扩增EV71病毒RNA,计算拷贝数,经10倍系列稀释做出标准曲线,作为EV71病毒RNA定量检测的外标准品。结果应用Qiagen公司QuantiTect SYBR Green RT-PCR Kit,该标准品可精确定量到100copies/μL,PCR扩增效率达到99.5%。结论 SYBRGreenⅠ荧光染料实时定量PCR法测定EV71病毒RNA拷贝数的方法敏感性高、稳定性好,可用于EV71病毒RNA载量的定量测定。     

应用SYBR实时荧光定量Real—TimePCR法检测人宫颈癌MCPH1 mRNA表达

目的 应用SYBR实时荧光定量RT-PCR法检测MCPH1/BRIT1 mRNA的表达.方法 提取人宫颈癌总RNA,经逆转录PCR获得靶基因（MCPH1）及管家基因（GAPDH）的CDNA,采用SYBR Green 荧光实时定量法检测,以GAPDH基因作为内参,计算各组MCPH1 mRNA的相对表达量.结果 在31例宫颈癌标本中,MCPH1基因mRNA的表达,19例癌比正常低,1 例癌比正常高,其余标本无统计学意义;6例癌比癌旁低,1例癌比癌旁高,其余无统计学意义.结论 利用SYBR实时荧光定量RT-PCR检测出人宫颈癌中MCPH1基因mRNA的表达下调,为进一步研究MCPH1在宫颈癌中的作用及功能奠定了基础.     

食品中阪崎肠杆菌快速检测鉴定方法的研究

通过设计特异性的引物,采用SYBR Green I实时荧光PCR,经引物的优化筛选、特异性和重现性试验,以及模拟污染样品检验,建立了食品中阪崎肠杆菌的快速检测和鉴定方法。     

荧光定量RT-PCR检测血管内皮细胞PAI-1 mRNA方法的建立

为了建立用荧光定量PCR检测人微血管内皮细胞纤溶酶原激活剂抑制物-1（PAI-1）mRNA表达的方法。提取人微血管内皮细总RNA,经RT-PCR获得靶基因（PAI-1）及管家基因（β-actin）的PCR产物。纯化后,作为标准品梯度稀释,采用SYBR Green I定量PCR检测,建立标准曲线。方法学考核参数为特异性、线性范围、灵敏性和重复性。分析全反式维甲酸对内皮细胞表达PAI-1 mRNA的干预效果。这种定量方法特异性好,检测的灵敏度达10^3拷贝,线性范围为10^4-10^10拷贝,循环阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系（r^2〉0.990）,批内变异≤4.93%,批间变异≤9.12%。全反式维甲酸能上调内皮细胞PAI-1 mRNA的表达,且呈剂量依赖性。因此,荧光定量RT-PCR检测血管内皮细胞PAI-1基因表达的方法有助于溶栓药物药理学的研究和新药筛选。     

RT－cDNA克隆－PCR法获取犬瘟热病毒融合蛋白基因（F）

纯化鸡胚成纤维细胞培养的犬瘟热病毒（ＣａｎｉｎｅＤｉｓｔｅｍｐｅｒＶｉｒｕｓ,ＣＤＶ）,获得病毒基因组ＲＮＡ后,反转录合成双链病毒Ｆ基因ｃＤＮＡ。将此双链ｃＤＮＡ平端插入ＰＵＣ１９质粒ＳａｍⅠ位点构建重组质粒,进行ｃＤＮＡ克隆。以重组克隆质粒为模板ＰＣＲ扩增,获得ＣＤＶ全长Ｆ基因。将此Ｆ基因插入表达载体ＰＢＶ２２０,在大肠杆菌中表达,通过对表达产物的最终鉴定,可确认所获片段为ＣＤＶ全长Ｆ基因．     

狐源犬瘟热病毒CDV-FOX-TA株核蛋白基因的原核表达及鉴定

应用RT-PCR技术扩增出犬瘟热病毒(CDV) 核衣壳(N)蛋白基因抗原性好的高保守基因片段,将其TA克隆至pMD18-T载体中,再利用酶切、连接的方法将测序正确的N基因目的片段亚克隆到原核表达载体pET24b中6×His Tag编码基因的上游,并将该重组质粒转化大肠杆菌Rosetta 2 (DE3)株,经IPTG诱导,N基因融合蛋白获得了高效表达。SDS-PAGE 分析和Western blot 分析的结果显示,表达产物的分子质量为15 kD,与CDV标准阳性血清呈阳性反应,间接ELISA结果也表明重组表达产物具有良好的抗原性,能够有效区分CDV标准阳性与阴性血清,表明大肠杆菌表达的CDV N 蛋白在免疫原性上具有与天然N蛋白同样的特性,可作为检测CDV的间接ELISA包被抗原。     

SYBR Green I and TaqMan quantitative real-time polymerase chain reaction methods for the determination of amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1)

The pfmdr1 gene, which encodes P-glycoprotein homolog 1, has been shown to be a reliable marker of resistance for Plasmodium falciparum related to artesunate and mefloquine combination therapy. The aims of this study are to investigate the copy number of pfmdr1 in P. falciparum isolates collected from the 4 malaria-endemic areas of Thailand (Kanchanaburi, Mae Hongson, Ranong, and Tak) along the Thailand-Myanmar (Burma) border (Thai-Myanmar border) by using SYBR Green I and the standard method TaqMan real-time polymerase chain reaction (RT-PCR) and to compare the efficiency (sensitivity and specificity) of SYBR Green I with TaqMan RT-quantitative (q)PCR methods in determining pfmdr1 gene copy number. Ninety-six blood samples were collected onto filter paper from patients with uncomplicated falciparum malaria who attended malaria clinics in the Kanchanaburi (n = 45), Mae Hongson (n = 18), Ranong (n = 11), and Tak (n = 22) provinces in Thailand. Parasite genomic DNA was extracted from dried blood spots by using QIAcube? automated sample preparation. Pfmdr1 gene copy number was determined by TaqMan (63 samples) and SYBR Green I (96 samples) real-time PCR. Seventy-one (74.0%), 14 (14.6%), 10 (10.4%), and 1 (1%) isolates carried 1, 2, 3, and 4 pfmdr1 gene copies, respectively. Forty-three of 48 (89.6%), 6 of 11 (54.5%), and 3 of 4 (75.0%) samples, respectively, showed agreement with results of 1, 2, and 3 pfmdr1 gene copies as determined by both methods. The efficiency of SYBR Green I in identifying pfmdr1 gene copy number was found to be significantly correlated with that of TaqMan. Considering its simplicity and relatively low cost, SYBR Green I RT-qPCR is therefore a promising alternative technique for the determination of pfmdr1 copy number.     

实时荧光定量PCR方法快速检测转基因大豆

针对转基因大豆中普遍含有的35S启动子进行引物设计,以双链DNA染料SYBR GreenⅠ为荧光标记物,利用实时荧光定量PCR方法对大豆样品进行检测。该法检测转基因大豆的检测低限为0.005 nmol/L的35S启动子,线性范围达3个数量级,可快速区分转基因大豆和非转基因大豆,具有快速、简便、灵敏、安全、高通量、低成本等优点,可推广用于转基因植物产品的快速定量检测。     

Detection of the Escherichia coli pathogenic gene eae with three real-time polymerase chain reaction methods

Several real-time polymerase chain reaction (PCR) methods are currently available to rapidly detect the presence of a specific DNA sequence. When used for detection of pathogenic organisms, the turnaround time for PCR-based methods is much lower than for traditional culture techniques. This study compared the sensitivity of three real-time PCR methods when detecting the Escherichia coli pathogenic gene eae to determine which method is most effective in identifying very low levels of the organism. The three methods were used to detect the eae gene over a range of DNA concentrations. The differences in sensitivity were statistically significant (p<0.05), and SYBR Green I PCR was found to have the lowest detection limit of the three; LUX primers had the highest detection limit. Therefore, using a defined DNA concentration for detecting the eae gene, SYBR Green I is the best alternative.     

Performance of Simplexa Dengue Molecular Assay Compared to Conventional and SYBR Green RT-PCR for Detection of Dengue Infection in Indonesia

Diagnostic tests based on detection of dengue virus (DENV) genome are available with varying sensitivities and specificities. The Simplexa Dengue assay (Focus Diagnostics) is a newly developed real-time RT-PCR method designed to detect and serotype DENV simultaneously. To assess the performance of the Simplexa Dengue assay, we performed comparison with conventional RT-PCR and SYBR Green real-time RT-PCR on patients sera isolated from eight cities across Indonesia, a dengue endemic country. A total of 184 sera that were confirmed using NS1 and/or IgM and IgG ELISA were examined. Using conventional and SYBR Green real-time RT-PCR, we detected DENV in 53 (28.8%) and 81 (44.0%) out of 184 sera, respectively. When the Simplexa Dengue assay was employed, the detection rate was increased to 76.6% (141 out of 184 samples). When tested in 40 sera that were confirmed by virus isolation as the gold standard, the conventional RT-PCR yielded 95% sensitivity while the sensitivity of SYBR Green real-time RT-PCR and Simplexa Dengue assay reached 97.5% and 100%, respectively. The specificities of all methods were 100% when tested in 43 non-dengue illness and 20 healthy human samples. Altogether, our data showed the higher detection rate of Simplexa Dengue compared to conventional and SYBR Green real-time RT-PCR in field/surveillance setting. In conclusion, Simplexa Dengue offers rapid and accurate detection and typing of dengue infection and is suitable for both routine diagnostic and surveillance.     

两种核酸染料染色效果的比较研究

用前染和后染两种不同的染色方法,研究比较SYBRGreenI和溴化乙锭(EB)两种核酸染料对凝胶中DNA的染色效果和灵敏度,及SYBRGreenI取代EB用于常规凝胶中核酸染色的可能性。结果表明,用前染法染色SYBRGreenI对琼脂糖凝胶中的核酸染色效果与EB相当;用后染法染色前者要优于后者,可显示5ng以下的DNA条带,在完全相同的操作条件下,其染色DNA条带背景清晰,灵敏度较高。因此,无致突变性新型染料SYBRGreenI可替代强致突变性染料EB用于检测凝胶中DNA片段大小、含量等,从而减少由于使用EB带来的环境污染和人体健康危害。