聚合酶键式反应生物芯片/微装置的实时定量荧光检测

以微电子机械系统技术而发展起来的聚合酶链式反应(Polymerase Chain Re-action,PCR)生物芯片/微装置,由于具有所需样品和反应混合物体积少,反应速度快以及集成化程度高等优点而日益引起人们的重视。实时定量PCR是利用能特异标记PCR产物的荧光染料来动态显示PCR产物的累积,从而得到PCR扩增曲线的技术。本文介绍了实时定量检测技术及其在PCR生物芯片/微装置中的应用。     

芝麻致敏原的实时荧光PCR检测

针对芝麻Sesamum indicum 2S albumin mRNA基因设计引物、探针,在实时荧光PCR仪上进行扩增、检测和分析.结果显示:该组探针和引物对芝麻有很强的特异性,除黑芝麻和白芝麻外,其余6种对照材料均未检测到荧光信号,黑白芝麻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,可用于芝麻致敏原成分的定量检测.     

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.     

聚乙二醇沉淀富集和预扩增反转录实时定量聚合酶链式反应高灵敏检测小浆果中低浓度甲型肝炎病毒

基于聚乙二醇沉淀富集和改进的预扩增反转录实时定量聚合酶链式反应(RT-qPCR),建立了高灵敏检测小浆果中低剂量感染甲型肝炎病毒(Hepatitis A virus, HAV)的方法。优化了聚乙二醇沉淀富集草莓中HAV的处理时间,探索设置预扩增步骤以加速样本核酸裂解和富集模板,建立了一种高灵敏的预扩增RT-qPCR检测新方法。回收率为15.51%, HAV减毒疫苗检测灵敏度可低至滴度4.49 CCID₅₀/mL,检测HAV质粒的灵敏度低至3.5 copies/μL。与ISO/TS15216-2:2019标准方法相比,本方法具有更高的检测效率,适于对小浆果中微量污染HAV的检测。对78批市售小浆果样品中HAV的阳性检出率为5.13%,高于采用ISO/TS 15216-2:2019标准方法的检出率(1.28%)。本方法对低剂量HAV病毒污染小浆果的检测具有很好的适用性和推广应用前景。     

基于微磁珠分离技术的适配体实时定量PCR检测方法

利用适配体的识别能力和可扩增性, 构建了基于微磁珠分离技术的适配体实时定量聚合酶链式反应(PCR)检测方法. 通过微磁珠偶联的互补链与适配体序列之间的碱基配对结合, 有效除去溶液中未与靶分子结合的适配体序列, 采用实时定量PCR技术测定上清液中结合态的适配体序列浓度, 从而间接实现对靶分子的定量检测. 分别选取代表生物大分子和有机小分子的凝血酶和ATP作为检测对象, 验证了该方法的普适性. 研究结果表明, 在获取特异性适配体序列后, 仅需简单优化其互补链序列, 即可对超低含量的凝血酶和ATP进行准确定量, 检出限分别为50 pmol/L和5 μmol/L. 该方法具有同时适用于高特异性和高灵敏度地检测生物大分子和有机小分子的优势.     

一种αⅠ型干扰素基因新变种的发现和鉴定

本文从一名中国汉族正常人胎肝细胞染色体DNA中,应用PCR方法两次获得长为520 bp的人α型干扰素基因,核苷酸序列测定表明,两次扩增产物的DNA序列完全相同,与过去分离克隆的IFN-αI和IFN-αD基因相比较,其第410位和第541位核苷酸分别为C和G,由此推测它编码的IFN成熟肽第114位和第158位氨基酸应为Ala和Val,其余位点则与IFN-αD和IFN-αI完全相同.我们建议将IFN—αD,IFN—αI和我们分离的IFN-αI/158V基因分别命名为IFN-αIa,IFN-αIb和IFN-alc基因.     

数字PCR在食品安全检测中的应用研究进展

聚合酶链反应(PCR)在动植物源掺杂鉴别、转基因成分和致病微生物等食品安全检测领域成为日趋重要的检测技术。从传统PCR、荧光PCR到数字PCR,PCR技术逐渐从定性分析、半定量分析发展到准确定量,不仅提升了准确度和检测效率,同时也扩展了食品检测范围,使食品安全的监管更加精细化。该文总结了近5年来数字PCR在食品安全检测中的研究进展,比较了传统PCR、荧光定量PCR和数字PCR的相关标准制订情况,列举、讨论了数字PCR在不同食品安全领域检测中的技术进展和存在的问题,并对数字PCR未来发展方向进行了展望。     

基于适配体的实时定量PCR检测血清中的人促红细胞生成素

基于适配体的高特异识别能力和可扩增性,构建了经磁分离后实时定量PCR检测重组人促红细胞生成素-α(rHuEPO -α)的新方法;针对实际血清样品中高丰度蛋白质等的干扰,利用碱基互补配对原理设计合成了分别与适配体两端引物区结合的互补链,通过凝胶迁移阻滞分析(EMSA)筛选出最佳的互补链,并将生物素化的互补链连接到链霉亲和素磁珠上,以此为探针捕获复杂基质中形成的待测复合物.研究结果表明,结合该样品前处理策略,该方法可成功地应用于正常人血清中的rHuEPO -α定量检测,检出限为25pmol/L,线性范围为50 pmol/L～ 50 nmol/L.     

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。     

Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.     

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.     

Quantitative Detection of Gene Methylated Level of Stool Samples Based on Invader Assay Coupled with Real-time Polymerase Chain Reaction and Its Application in Non-invasive Screening of Colorectal Cancer

Methylation of bone morphogenetic protein 3 (BMP3) in stool DNA is an effective biomarker for non-invasive screening of colorectal cancer. However, a highly sensitive and specific detection method is required. Here, a quantification method for BMP3 methylation was developed by combining real-time polymerase chain reaction (PCR) with invader assay using Beta-actin (ACTB) as a reference. Amplification efficiencies of BMP3 and ACTB were close to 100% after optimizing the concentration of detection probes, FEN1 enzyme and Taq polymerase, and the relative quantification of BMP3 methylation was achieved accurately by ΔCT algorithms. Ten copies and 0.01% of BMP3 methylation level could be successfully detected and non-specific signal was generated from non-methylated template, indicating that the method was highly sensitive and specific. The method was successfully applied to detect BMP3 methylation in fecal DNA from 16 colorectal cancer patients, 7 adenoma patients and 19 healthy volunteers. The results indicated that BMP3 methylation occurred in 5 of 16 cancer patients and 2 of 7 adenoma patients, but was not observed in 19 of healthy volunteers. Therefore, this method could be used to quantify methylation of gene in stool samples, providing an effective technique for non-invasive screening of colorectal cancer.     

一种用于核酸绝对定量检测的高鲁棒性液滴式数字PCR芯片

为满足液滴式数字聚合酶链式反应(PCR)技术对扩增反应过程中稳定保存液滴以及反应后高效检测的核心需求,构建了一种具有过滤气泡和增强荧光信号功能的液滴式数字聚合酶链式反应芯片.该芯片可在10 min内产生20多万个半径约为21μm的液滴.利用"玻璃天花板"的方式构建了独立于芯片主体材料的液滴收集腔,为液滴提供稳定的保存与反应环境;还构建了过滤结构,可有效过滤混入液相中的空气,提高芯片鲁棒性.同时,在液滴收集腔中引入反射层,增强荧光信号,使单个视野荧光成像时间缩短约40%,提高了检测效率.利用该芯片定量检测EGFR基因第21号外显子,检测信号与DNA浓度在10¹～10⁵copies/μL范围内呈现良好的线性关系(R²=0.998).该方案在载玻片大小的芯片上实现了液滴产生、PCR扩增和荧光信号读取,并具有较高的鲁棒性与检测效率,在核酸检测等方面具有应用潜力.     

实时荧光聚合酶链式反应偶联高特异性核酸侵入反应检测单核苷酸多态性

建立了实时荧光聚合酶链式反应( PCR)偶联高特性核酸侵入反应检测单核苷酸多态性( SNP)的方法。优化了体系中flap核酸内切酶1(FEN1酶)和野生型检测探针等用量,确定了最佳反应条件,即FEN1酶用量为1.5 U,野生型检测探针用量为0.125μmol/L,0.5μmol/L Invader突变型检测探针,各0.25μmol/L通用野生型( VIC)和突变型( FAM)荧光共振转移发卡探针,显著降低了野生型样本和突变型样本背景信号,避免了背景信号对检测结果分型的干扰。采用本方法对编码乙醛脱氢酶2( ALDH2)基因ALDH2*2位点21例样本、细胞色素P4502C19基因CYP2C19*2和CYP2C19*3位点各19例样本进行分型检测,结果表明, AL-DH2*2位点GG纯合10例,GA杂合8例,AA纯合3例;CYP2C19*2位点GG纯合9例,GA杂合8例,AA纯合2例;CYP2C19*3位点GG纯合18例,GA杂合1例。使用焦磷酸测序进行验证,两种方法检测结果一致。本方法特异性好、操作简便、耗时短、成本低,可实现对SNP单管闭管无污染的分型检测。     

荧光分析法在聚合酶链式反应产物检测中的应用

对荧光法在聚合酶链式反应（ＰＣＲ）产物检测中的应用作了评述。内容包括原理、方法和仪器,并对今后找作进行了展望。     

Transformation of arsenic(V) by the fungus Fusarium oxysporum melonis isolated from the alga Fucus gardneri

A fungus isolated from the macroalga Fucus gardneri was identified by using 28S rDNA sequence analysis, 99% similarity match, as Fusarium oxysporum meloni. The fungus was exposed to arsenic(V) (500 ppb) in artificial seawater to investigate the possibility that the fungus is the source of the metabolic activity that results in the presence of arsenosugars in the macroalga. High‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry was used to identify the arsenic species in the fungus, and in the growth medium. The fungus was able to accumulate arsenic(V) and an increase in arsenite and dimethylarsinate was also observed. Some reduction of arsenate led to a small increase of arsenite in the growth medium. The fungus does not seem to be involved with the accumulation of arsenosugars by the Fucus. Copyright © 2002 John Wiley & Sons, Ltd.     

电化学发光PCR定量检测H-ras癌基因点突变

提出了一种电化学发光PCR(ECL-PCR)分析方法,该法可用于定量检测基因点突变.其检测H-ras癌基因PCR扩增产物的灵敏度可达100fmol;线性范围为0.1-500pmol.用ECL-PCR分析法对膀胱癌组织中H-ras癌基因进行突变检测,只需要10μL样品,20min的孵育时间和30s的采集时间,得出20例膀胱癌样品中有7例存在点突变,通过标准曲线方程定量计算出突变样品的量.ECL-PCR分析方法在灵敏度、线性范围、分析时间等方面都优于传统的检测方法,是一种安全、快速、灵敏、定量检测基因点突变的分析方法.     

Detection of BCR- ABL Gene Rearrangement by RT/PCR Technology and Its Mechanism in the Generation and Development of Chronic Myeloid Leukemia

ＩｎｔｒｏｄｕｃｔｉｏｎＣｈｒｏｎｉｃＭｙｅｌｏｉｄｌｅｕｋｅｍｉａ（ＣＭＬ）ｗａｓａｍａｉｎｄｉｓｅａｓｅｓｈｏｗｎｔｏｂｅｃｏｎｓｉｓｔｅｎｔｌｙａｓｓｏｃｉａｔ－ｅｄｗｉｔｈｔｈｅｃｙｔｏｇｅｎｅｔｉｃａｂｎｏｒｍａｌｉｔｙｎｏｗｋｎｏｗｎａｓｔｈｅＰｈｉｌａｄｅｌｐｈｉａ（Ｐｈ′）ｃｈｒｏｍｏｓｏｍｅ．Ｔｈｅａｂｎｏｒｍａｌｉｔｙｕｓｕａｌｌｙｉｎｖｏｌｖｅｓａｒｅｃｉｐｒｏｃａｌｔｒａｎｓｌｏｃａｔｉｏｎｂｅｔｗｅｅｎｃｈｒｏｍｏｓｏｍｅｓ９ａｎｄ２２,ａｎｄｍｏｒｅｔｈａｎ９５％ｏｆ…     

Kinetics of abiotic and biotic degradability of low-density polyethylene containing prodegradant additives and its effect on the growth of microbial communities

The kinetics of abiotic oxidation in the dark and the kinetics of biological mineralization in soil and in a compost environment of thermally oxidized LDPE were studied. It was demonstrated that different activation energies are obtained for the thermal oxidation, depending on the composition of the materials. Significantly higher levels of biodegradability have been obtained in a soil environment at 23 °C compared with the compost environment at 58 °C. After two years of mineralization, 91% conversion to carbon dioxide was obtained in the soil test, compared with 43% in the compost test. The differences between fungal, archaeal and bacterial community structures in soil and compost after 607 days of biodegradability assay were mapped out. It was found that the most dominant bacterial and fungal terminal restriction fragments (TRFs) in the compost containing the test material are significantly different from the TRFs in the other environments.