复合式蝎形引物实时定量检测端粒酶延伸产物

针对端粒酶延伸产物中靶基因序列的特殊性,开发了一种可产生荧光的复合式蝎形引物,该引物的5'端带有可特异性检测靶基因的探针序列,PCR阻断剂将其与引物序列连接.当复合式蝎形引物延伸,探针序列与同一分子内的靶基因杂交,荧光信号产生.运用该技术,建立了定量检测端粒酶延伸产物的实时荧光PCR方法.该法可在快速PCR循环条件下,对0.15～1.50×10³ amol/μL范围内的样品进行定量检测,线性相关系数R²=0.9992.该法操作简便,无需PCR后额外的检测步骤.     

Taqman-MGB实时荧光PCR定量检测转基因玉米MON863

运用实时荧光聚合酶链式反应技术(polymerase chain reaction,PCR)对转基因玉米MON863进行品种特异性检测和定量分析。通过设计玉米内源基因和外源基因边界序列特异性引物和Taqman-MGB探针,验证了内源基因的物种特异性和外源基因边界序列的品种特异性。利用已知转基因百分含量的MON863玉米作为标准品,进行荧光定量反应,建立定量标准曲线,通过标准曲线对玉米样品MON863玉米成份进行含量分析。结果表明,该方法重复性好,检测特异性强,最低检测限浓度达到0.001 ng/μL,即14个拷贝。由于使用实时荧光PCR技术,检测周期短,操作简便,可广泛运用于转基因玉米MON863的进出口检测和转基因产品的含量分析。     

一种快速鉴别食用油的气相色谱指纹法

本发明公开了提供一种实时荧光定量PCR检测HC．MVgBn的分型方法,在PCR反应体系中加入荧光探针,利用荧光信号积累实时监测PCR进程,进行定量定性分析确定HCMVgBn型。本发明封闭反应,无需PCR后处理,避免污染;特异性强,灵敏度高采用对数期分析,摒弃终点数据,定量准确;实时荧光定量PCR技术既可对HCMVgB进行分型,又可对其进行定量;仪器在线式实时检测,结果直观,避免人为判断;可实现一管双检或多检;操作安全,缩短时间,     

牛奶α-乳白蛋白基因实时荧光定量PCR检测方法的建立

采用Taqman探针技术,建立食品中牛奶过敏原α-乳白蛋白基因的实时荧光定量PCR检测方法。根据α-乳白蛋白基因序列设计特异性引物及Taqman探针进行PCR扩增,构建质粒,经酶切鉴定测序后,建立拷贝数(copies)-Ct标准曲线。成功克隆α-乳白蛋白目的基因,建立的标准曲线在1.12×103~1.12×108 copies范围内线性关系良好,灵敏度高,液体样品检测限达到1 000copies/mL,特异性强,稳定性好。该法可用于实际商品中牛奶过敏原α-乳白蛋白组分的定量检测。     

目视化乙肝病毒基因芯片

优化了基于纳米金标记探针的目视化检测方法,将其用于检测从阳性血清中提取的乙肝病毒(HBV)基因,并与基于荧光素异硫氰酸酯(FITC)标记探针的荧光检测方法进行了比较.结果表明,目视化乙肝病毒基因芯片诊断方法操作简单,成本低廉.此类基因芯片在病毒基因检测领域将会有广泛用途.     

实时荧光等位基因特异性扩增法快速检测K-ras癌基因点突变

将荧光定量PCR技术与等位基因特异性扩增(Allele specific amplification, ASA)方法相结合, 发展了一种可以快速检测基因点突变的实时荧光等位基因特异性扩增(Real-time ASA)方法. 将该法用于检测K-ras癌基因第12位密码子发生的点突变, 分别采用针对其不同点突变方式(GAT, GTT, CGT)设计的突变型引物对待测样品进行ASA, 只有突变型样品能被顺利扩增出双链DNA产物, 该产物才能与双链DNA染料SYBR Green Ⅰ结合, 发出荧光信号从而被检测到. 用该法检测31例结肠癌组织中的K-ras癌基因点突变, 其中有15例样品检出为突变型. Real-time ASA法可检测到样品中含量为1/1000的突变型基因, 具有灵敏、快速、简便、安全、高通量和低成本等优点, 可望用于大量临床样本的点突变筛查.     

污染水产去甲基化表观遗传毒性EGFP评价方法

pEGFP-C3质粒经过体外人工甲基化处理后,被转染进入HepG2细胞以构建重组细胞株．以5-AZA为阳性去甲基化毒物与重组细胞共培养,通过亚硫酸氢钠测序法定量检测EGFP基因启动子区甲基化状态,通过实时定量PCR检测EGFP基因表达,借助流式细胞术和荧光摄片定量检测共培养细胞的绿色荧光强度,在DNA甲基化、EGFP基因mRNA表达、GFP蛋白等多个层次研究5-AZA染毒处理与其去甲基化能力和荧光表达改变的响应关系．对天津污染水产的去甲基化能力进行了实际样品测试．结果表明,5-AZA与重组细胞的DNA甲基化、基因表达、蛋白产物变化之间存在显著关联,具有较低的检出浓度和良好的重复性．天津污染海域的水产去甲基化能力较强．本文初步建立了一种污染物去甲基化表观遗传毒性评价方法．     

用于高度降解mRNA样本的非反转录基因表达量测定方法

许多生物样本(如石蜡包埋组织样本)中的mRNA易断裂为小片段,利用传统方法检测较困难。为了测定高度降解的mRNA,本研究针对待测mRNA短片段设计一对探针,当探针与待测模板杂交后,通过连接反应将两条探针5’与3’端相连,连接产物作为PCR扩增模板进行实时荧光定量检测,从而对待测mRNA进行定量测定。以人ACTB基因为待测靶标,通过测定不同浓度的待测靶标及与待测靶标序列不同的RNA片段,分别考察方法的灵敏度与特异性,并检测肺癌石蜡切片样本中ACTB基因的表达量,与传统的反转录定量PCR检测结果进行了对比。本方法的检出限为150 fmol/L,定量线性范围为150 fmol/L～300 pmol/L,并且具有良好的特异性。在对石蜡包埋组织样本中的基因表达量检测时,本方法扩增检测CT值比反转录实时定量PCR小,表明本方法更适合对高度降解的mRNA样本进行定量测定。     

巴西果仁致敏原的实时荧光PCR检测

针对巴西果仁2Salbumin mRNA基因设计引物、探针,在实时荧光PCR仪上进行扩增、检测和结果分析.结果显示:该组探针和引物对巴西果仁有很强的特异性,除巴西果仁外,其余8种对照树果材料均未检测到荧光信号,巴西果仁成分检测灵敏度达到0.1%.该方法具有灵敏度高、快速、简便的特点,可用于巴西果仁致敏原成分的定量检测.     

转基因玉米的多重PCR-毛细管电泳-激光诱导荧光检测方法研究

采用三重PCR反应, 同时扩增CaMV 35S启动子、 hsp70 intron1和CryIA(b)基因之间序列以及Invertase基因, 扩增产物用无胶筛分毛细管电泳-激光诱导荧光检测, 从而建立了多重PCR-毛细管电泳-激光诱导荧光快速检测转基因玉米的新方法. 对影响多重PCR扩增和毛细管电泳的因素进行了优化. 在优化的条件下, 本方法可以同时检测转基因玉米样品中3种外源基因. 经序列测试证实, 三重PCR 扩增产物的序列与原基因完全一致, 表明扩增结果可靠. 该方法能检出0.05% MON810转基因玉米成分, 远低于欧盟对转基因食品规定标识的质量分数阈值(1％). 该方法对玉米及其制品的检测结果与实时荧光PCR方法的检测结果一致, 与传统的琼脂糖凝胶电泳法相比, 具有特异性高\, 快速及灵敏等优点, 适用于玉米中转基因成分以及转基因玉米MON810品系的快速筛选、 鉴定和检测, 能满足我国实施转基因食品标签法规的要求.     

利用无标记的分子信标及核酸染料SYBR Green Ⅰ检测乙肝病毒

本文以野生型的乙型肝炎病毒(HBV)核酸片段为研究对象,利用无标记的分子信标及核酸染料SYBR Green I,建立了一种高灵敏、高选择性的特定序列核酸检测方法.在优化条件下,目标DNA浓度为4×10-11~400×10-11 mol/L之间时,SYBR Green I的荧光强度(ΔI)与目标DNA的浓度(C)具有良好的线性关系,其拟合的回归方程为ΔI=1.9556 C+31.4659(R2=0.9956),方法检测限(3ζ)为2×10-11 mol/L.该方法操作简单、检测速度快、灵敏度高、重现性好、检出限低.利用该方法,结合不对称PCR技术,实现了对HBV的定量检测.     

Quick genotyping detection of HBV by giant magnetoresistive biochip combined with PCR and line probe assay

Genotyping of human hepatitis B virus (HBV) can be used to direct clinically effective therapeutic drug-selection. Herein we report that a quick genotyping method for human HBV was established by a specially designed giant magnetoresistive (GMR) biochip combined with magnetic nanoclusters (MNCs), PCR and line probe assay. Magnetic nanoclusters of around 180 nm in diameter were prepared and modified with streptavidin, and resultant streptavidin-modified magnetic nanoclusters were used for capturing biotin-labeled hybrid products on the detection interface of the sensor. The gene fragments of HBV's B and C gene types were obtained by PCR based on a template of B- and C-type plasmids. After gene fragments were hybridized with captured probes, streptavidin-modified magnetic nanoclusters could bind with biotin-conjugated gene fragments, and the resultant hydride products could be quickly detected and distinguished by the GMR sensor, with a detection sensitivity of 200 IU mL(-1) target HBV DNA molecules. The novel method has great potential application in clinical HBV genotyping diagnosis, and can be easily extended to other biomedical applications based on molecular recognition.     

Compressed Air-Driven Continuous-Flow Thermocycled Digital PCR for HBV Diagnosis in Clinical-Level Serum Sample Based on Single Hot Plate

We report a novel compressed air-driven continuous-flow digital PCR (dPCR) system based on a 3D microfluidic chip and self-developed software system to realize real-time monitoring. The system can ensure the steady transmission of droplets in long tubing without an external power source and generate stable droplets of suitable size for dPCR by two needles and a narrowed Teflon tube. The stable thermal cycle required by dPCR can be achieved by using only one constant temperature heater. In addition, our system has realized the real-time detection of droplet fluorescence in each thermal cycle, which makes up for the drawbacks of the end-point detection method used in traditional continuous-flow dPCR. This continuous-flow digital PCR by the compressed air-driven method can meet the requirements of droplet thermal cycle and diagnosis in a clinical-level serum sample. Comparing the detection results of clinical samples (hepatitis B virus serum) with commercial instruments (CFX Connect; Bio Rad, Hercules, CA, USA), the linear correlation reached 0.9995. Because the system greatly simplified the traditional dPCR process, this system is stable and user-friendly.     

Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection

A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence. The U-MB technique not only reduced the cost of HBV detection but also had the potential for the development of a universal detection kit for different mutant HBV types and other DNA systems. To demonstrate its clinical utility, 90 serum samples were analyzed using the U-MB real-time PCR method. In the experiments we found that several crucial factors needed to be considered in the primer design, such as the avoidance of formation of severe primer–dimer and primer self-hairpin structure. With the optimized primer sets, satisfactory results were obtained for all the tested samples. We concluded that this assay would be an excellent candidate for a universal HBV DNA detection method. Principle of the U-MB real-time PCR method for HBV DNAdetection     

ssDNA-QDs/GO multicolor fluorescence system for synchronous screening of hepatitis virus DNA

Viral hepatitis is a common infectious disease caused by five viruses (hepatitis virus A, B, C, D, and E). Given the diversity of hepatitis virus, rapid screening and accurate typing of viral hepatitis are the prerequisites for hepatitis therapy. Here, a multicolor fluorescence system was constructed by combining with the multi-color fluorescence properties of CdSe/ZnS quantum dots (QDs, emission wavelengths: 525 nm, 585 nm and 632 nm) and the broad-spectrum fluorescence quenching performance of GO. Taking advantage of the specific recognition of ssDNA modified CdSe/ZnS QDs to target hepatitis virus DNA, the constructed system could effectively distinguish hepatitis A virus DNA (HAV-DNA), hepatitis B virus DNA (HBV-DNA), and hepatitis C virus DNA (HCV-DNA) in a homogeneous solution. Based on the different adsorption property of GO for ssDNA and dsDNA, the fluorescence Forster resonance energy transfer (FRET) process between ssDNA modified QDs and GO could be regulated. The fluorescence signal of the constructed system presented a sensitive response to HAV-DNA, HBV-DNA, and HCV-DNA content in the range of 1.0–192 nM, 8.0–192 nM, and 1.0–128 nM, respectively. The limit of detection for HAV-DNA, HBV-DNA, and HCV-DNA is 0.46 nM, 1.53 nM, and 0.58 nM. The constructed system can be used to screen hepatitis virus DNA in real samples, which provides an alternative strategy for rapid screening and diagnosis of viral hepatitis.     

Application of Single—labelled Probe—primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

A new method based on the incorporation of a single-lablled probe-primer into polymerase chain reaction(PCR) for the detection of PCR-amplified DNA in a closed system is reported.The probeprimerc consists of a specific probe sequence on the 5‘‘‘‘‘‘‘‘-end and a primer sequence on the 3‘‘‘‘‘‘‘‘-end.A flurophore is located at the 5‘‘‘‘‘‘‘‘end.The primeR-quencher is an oligonucleotide,which is complementary to the probe sequence of probe-primer and labelled with a quencher at the 3‘‘‘‘‘‘‘‘-end.In the duplex formed by probe-primer and primer-quencher.the fluorophore and quencher are kept in close proximity to each other.Therefore the fluorescence is quenched.During PCR amplificatio,the specific probe sequence of probeprimer binds to its complement within the same strand of DNA,and is cleaved by Taq DNA polymerase,resulting in the restoration of fluorescence.This system has the same energy transfer mechanism as molecular beacons,and a good quenching effciency can be ensured.Following optimization of PCR conditions,this method was used to detect hepatitis b virus(HBV) dna in patient sera.This technology eliminates the risk of carry-over contamination,simplifies the amplification assay and opens up new possibilities for the real-time detection of the amplified DNA.     

Validation of a Reference Gene (BdFIM) for Quantifying Transgene Copy Numbers in Brachypodium distachyon by Real-Time PCR

Brachypodium distachyon has been proposed as a new model system for gramineous plants with a sequenced genome and an efficient transformation system. Many transgenic B. distachyon plants have been generated in recent years. To develop a reliable fast method for detecting transgenic B. distachyon and quantifying its transgene copy numbers, a species-specific reference gene is of great priority to be validated both in qualitative PCR and quantitative real-time PCR detection. In this study, we first proved that the BdFIM (B. distachyon fimbrin-like protein) gene is a suitable reference gene in qualitative PCR and quantitative real-time PCR for B. distachyon. Fourteen different B. distachyon varieties were tested by both qualitative and quantitative PCRs, and identical amplification products of BdFIM were obtained with all of them, while no amplification products were observed with samples from 14 other plant species, suggesting that BdFIM gene was specific to B. distachyon. The results of Southern blot analysis revealed that the BdFIM gene was low copy number in seven tested B. distachyon varieties. In conclusion, the BdFIM gene can be used as a reference gene, since it had species specificity, low heterogeneity, and low copy number among the tested B. distachyon varieties. Furthermore, the copy number of inserted sequences from transgenic B. distachyon obtained by real-time PCR methods and Southern blot confirmed that the BdFIM gene was an applicable reference gene in B. distachyon.     

A Fluorescence Assay Based on GoldMag-CS Nanoparticles for Hepatitis B Virus DNA

A sensitive fluorescence assay for hepatitis B virus (HBV) DNA was developed based on the dissociation of bio-bar-code DNA probes from GoldMag-CS nanoparticles (NPs) and magnetic separation. In this method, the target sequence (HBV DNA) was recognized through sandwich hybridization by the catching probes and the detection probes. Catching probes were modified with biotins, and were specifically bound on streptavidin-coated 96-well microplates; detection probes were all attached on the GoldMag-CS nanoparticles, which also bound bio-bar-code strands with fluorescent tags. Bio-bar-codes were dissociated from the NPs by dithiothreitol (DTT) after DNA target recognition and magnetic separation, and then quantified. Streptavidin-coated 96-well microplates diminished the nonspecific binding of DNA-conjugated GoldMag-CS nanoparticles, thus lowering the background; and GoldMag-CS nanoparticles provided easy separation and significant signal amplification. Together, these two effects brought about the detection limit as low as 7.52 fM.