马铃薯立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测与应用

【背景】植物根际促生菌(plant growth-promoting rhizobacteria,PGPR)在根际的定殖是其发挥作用的基础,直观有效的跟踪技术和定量方法是研究PGPR在根际原位分布规律的重要工具。【目的】建立一种马铃薯黑痣病病原菌——立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测体系,并检测拮抗菌QHZ11在马铃薯根际的动态变化。【方法】根据GenBank中登录的类芽孢杆菌及近源菌株gyrB基因序列差异筛选特异性引物,优化反应条件;通过盆栽试验对马铃薯根际拮抗菌进行快速检测。盆栽试验设3个处理,T1:对照(无菌水,CK);T2:QHZ11菌悬液灌土(QHZ11);T3:将功能菌在有机肥中进行二次固体发酵制成生物有机肥(BOF11)。【结果】筛选出拮抗菌QHZ11的专用引物为gyrB-F/gyrB-R;建立的拮抗菌QHZ11实时荧光定量PCR检测方法特异性好、灵敏度高且重复性较好,线性相关系数为0.999 8,检测组内变异系数均在1%以内,扩增效率为0.9,可检测出1×103-1×1010copies/g-soil的拮抗菌,具有检出限低和扩增效率高的特点。盆栽试验...     

实时定量PCR法检测生物技术药物中宿主基因组DNA残留

利用基于SYBR GreenⅠ荧光染料的实时定量PCR方法检测酵母表达生物技术药物产品中宿主DNA残留量。该方法检测灵敏度可达到1.0 fg/μL, DNA浓度在1.0 fg/μL～1.0 ng/μL范围内线性良好,其标准曲线的相关系数为099以上。应用该方法对3批不同实验样本进行测定,宿主DNA残留量分别为8.635×105 fg/μL、6.265×102 fg/μL和1436 fg/μL 。实验表明该方法操作简便、灵敏度高,可用于生物技术药物产品中酵母DNA残留的定量测定。     

用实时定量PCR解决基因组序列组装中的重复序列问题

目的:探寻一种简单、经济的方法,解决基因组序列拼接中的重复序列问题。方法:选取序列拼接中遇到重复序列问题的质粒NDM-BTR,在其与重复序列相关的contigs两端设计引物,进行实时定量PCR,通过观察临界循环数来判断contig之间的位置关系。结果:成功判断出质粒contig之间的位置关系,得到了质粒基因组完成图。结论:实时定量PCR法可用于解决基因组序列拼接中的重复序列问题,相比较传统建立大片段文库更加简单、快速、经济。     

A组轮状病毒实时定量PCR检测方法的研究进展

A组轮状病毒是引起婴幼儿急性肠胃炎的最主要病原之一。目前已建立的轮状病毒检测方法有电镜检测、酶免疫法、反转录PCR法、实时定量PCR法。实时定量PCR法与其他方法相比具有特异性强、敏感度高、可以分型、定量准确等优势。本文就实时定量(Real-time)PCR技术应用于A组RV检测的研究进展进行简单概述。     

实时荧光定量PCR法检测转基因小鼠拷贝数

目的利用实时荧光定量PCR法鉴定转基因小鼠外源基因插入拷贝数。方法以TG-CARK转基因首见鼠为研究对象,选取小鼠的高度保守基因Fabpi为内参,利用绝对定量的实时荧光PCR法鉴定转基因小鼠拷贝数,并与传统的Southern blot方法的定量结果进行比较。结果实时定量PCR鉴定的转基因拷贝数与Southernblot法完全一致,三只TG-CARK首见小鼠的拷贝数分别为1,7,45。结论实时定量PCR技术具有高准确性、高稳定性、高通量和低成本的优点,是比传统杂交技术更好的鉴定小鼠转基因拷贝数的方法。     

土壤中立枯丝核菌AG3菌核的荧光定量PCR快速检测

立枯丝核菌Rhizoctonia solani融合群3(anastomosis group 3,AG3)是引发马铃薯黑痣病的主要病原菌,严重威胁着马铃薯产业的发展。菌核是马铃薯黑痣病菌在土壤中的主要存活结构,是马铃薯黑痣病的初侵染源,其密度直接影响马铃薯黑痣病的发病率和严重程度。为快速准确地检测出土壤中菌核的密度,本研究以立枯丝核菌AG3转录间区(ITS)特异性引物RsTqF1/RsTqR1扩增产物的重组质粒为标准品,构建SYBR Green I实时定量PCR(q PCR)检测体系,结合水筛法与Fast DNA?Spin Kit for Soil土壤DNA提取试剂盒对土壤样品进行DNA提取,建立了循环域值(Ct)与土壤中立枯丝核菌AG3菌核密度的关系,并与传统选择性培养基筛选法进行比较。结果表明,实时定量PCR检测灵敏度比常规PCR高10倍,土壤中菌核密度n(g/g土)和Ct值之间的关系为:n=10(9.6917‐Ct)/3.4558。相比较于选择性培养基筛选法,水筛q PCR法能够更加快速准确地检测出土壤中立枯丝核菌的菌核密度。     

实时荧光定量PCR相对定量法检测循环miRNA的内参选择

microRNA(miRNA)是一类内源性非编码微小RNA,在调控细胞增殖、分化、凋亡等方面发挥重要作用。研究发现,miRNA能够稳定存在于细胞外液,被称为循环miRNA。在多种疾病状态下循环miRNA表达谱改变,具有成为非创伤性新型生物标志物的潜能。实时荧光定量PCR(RT-qPCR)是检测miRNA表达变化的确证实验,其中内参的选择关系到相对定量结果是否真实可靠。我们简要综述国内外实验室常用的RT-qPCR内参及其特点。     

实时定量PCR方法检测鼻咽癌病人全血EB病毒拷贝数的实验研究

EBV在多种肿瘤中存在,并在相关肿瘤的发病中起一定的作用。本文应用实时定量PCR方法检测鼻咽癌与健康对照全血EBV拷贝数。结果表明在73例鼻咽癌病人与83例正常对照中,NPC组中EBV阳性率为46.6%,对照组阳性率为13.3%。对照组平均EB病毒拷贝数为3.9×10~4拷贝/μgDNA,高于鼻咽癌组(1.7×10~5拷贝/μgDNA)。全血EBV的感染与鼻咽癌的发生相关,而裂解状态EBV在细胞转化过程中的作用可能更大。应用实时定量PCR进行全血EBV的检测可用于鼻咽癌的分子诊断。     

实时荧光定量PCR 法用于重组SIV-hPEDF注射液病毒颗粒数的检测

目的:建立重组SIV-h PEDF注射液病毒颗粒数检测实时定量PCR检测方法。方法:在提取重组SIV-h PEDF注射液供试品基因组RNA后,采用实时定量PCR的方法,检测供试品中的病毒颗粒数。结果:经过3次实验,所得SIV-h PEDF RNA标准品标准曲线均线性良好,重组SIV-h PEDF注射液供试品中病毒颗粒数检测结果无显著性差异。结论:所建立的重组SIV-h PEDF注射液病毒颗粒数实时荧光定量PCR检测方法重复性好、灵敏、准确,可用于重组SIV-h PEDF注射液病毒颗粒数的检测。     

番茄叶片基因组DNA快速制备技术及其在基于实时荧光定量PCR的转基因检测中的应用

以番茄(Solanum lycopersicum L.cv.Micro-Tom)叶片为试材,建立了一种简便快速制备叶片基因组DNA的方法。2~20 mm2的叶片即可满足制备要求,制备过程只需一种提取试剂、只涉及1次移液和1次离心操作,不涉及沉淀。确定了所制备的DNA用于实时荧光定量PCR的合适用量为0.1~0.2μL(反应总体积为12.5μL),发现过量模板的使用可降低PCR效率且可导致扩增失败。该项DNA快速制备及相适应的实时荧光定量PCR技术已成功应用于番茄转基因植株检测。     

Genome size estimation with quantitative real‐time PCR in two Tephritidae species: Ceratitis capitata and Bactrocera oleae

The medfly Ceratitis capitata and the olive fruit fly Bactrocera oleae belong to the Tephritidae family of Diptera, a family whose members cause severe damages in agriculture worldwide. For such insect pests, the utmost concern is their population control. The sterile insect technique (SIT) has been used in the Tephritidae family with varying degrees of success. Its efficient use usually depends on the development of genetic sexing strains and the release of only male flies. However, such advances are based on modern genetic, molecular and genomic tools. The medfly is clearly the prototype of the family, since such tools have advanced considerably, which has resulted in effective SIT efforts around the world. A whole‐genome sequencing project of this insect is already underway. In contrast, similar tools in the olive fly lag behind, even though the insect is considered a promising candidate for a next SIT target. An accurate estimate of genome size provides a preliminary view of genome complexity and indicates possible difficulties in genome assembly in whole‐genome projects. Taking advantage of a quantitative real‐time PCR approach, we determined the genome size of these two species C. capitata and B. oleae as 591 Mb (CI range: 577–605 Mb) and 322 Mb (CI range: 310–334 Mb) respectively.     

转PvPGIP2基因小麦的获得与纹枯病抗性鉴定

多聚半乳糖醛酸酶抑制蛋白(PGIP)是一种植物防卫蛋白,可阻止一些病原真菌的侵害。本研究克隆出扁豆PvP-GIP2基因编码序列,构建了受玉米泛素(ubiquitin)启动子控制的PvPGIP2基因表达载体pA25-PvPGIP2;采用基因枪法将pA25-PvPGIP2转化小麦推广品种扬麦18幼胚愈伤组织4000块,获得了203株再生植株。PCR检测出阳性植株65株,转化率为1.625%。对转PvPGIP2基因小麦T1～T2植株,进行外源基因的PCR、RT-PCR、荧光定量RT-PCR(Q-RT-PCR)分析和小麦纹枯病抗性鉴定。结果表明,转入的PvPGIP2能够在转基因小麦中遗传、转录与表达;PvPGIP2基因的表达提高了转基因植株对小麦纹枯病的抗性。     

An improved method for determination of gene copy numbers in transgenic mice by serial dilution curves obtained by real-time quantitative PCR assay

Precise characterization of transgene insertion is necessary for phenotype interpretation of transgenic animals. To check for the presence of deletions, estimate the number of inserted transgene copies, and in addition, identify the zygosity of transgenic mice, gene copy numbers were determined by real-time quantitative PCR. Instead of correlating tested samples to a single relative standard curve, serial dilution curves were constructed for every mouse sample. A novel statistical approach was designed in which mice with the same copy number were characterized by the adjusted group mean and standard deviation common to the target sequence. This enabled us to characterize the variability of the obtained results, statistically compare different groups of mice and estimate precision and limits of the applied method.     

实时荧光定量PCR(TaqMan)法测定外源基因的拷贝数

实时荧光定量PCR是近年新兴的一项技术,因其快速、方便、便宜,需要DNA样品量少,无需放射性检测等优点被广泛应用于基因的定量分析。该文就实时荧光定量PCR(TaqMan)技术的发展、基本原理及测定外源基因拷贝数的技术流程做一介绍。     

Genome size estimation of liver fluke Opisthorchis viverrini by real-time polymerase chain reaction based method

The human liver fluke, Opisthorchis viverrini, has been categorized as a class one carcinogenic organism according to its strong association with cholangiocarcinoma, bile duct cancer which has high incidence in the northeast of Thailand. The lack of genome database of this parasite limited the studies aimed to understand the basic molecular biology of this carcinogenic liver fluke. The determination of the genome size is an initial step prior to the full genome sequencing. In this study, we applied an absolute quantitative real-time polymerase chain reaction for this aspect. Our results indicated the genome size of O. viverrini is 75.95 Mb or C value 0.083. The information of O. viverrini genome size is useful for estimation of sequence coverage and the cost of the parasite's whole genome sequencing using next-generation sequencing technologies.     

基于实时定量PCR技术的育龄期细菌性阴道病患者阴道菌群结构分析及其临床应用

目的 探讨育龄期女性细菌性阴道病（bacterial vaginosis,BV）患者阴道优势菌群变化及其与临床指标的相关性。方法 选择本院门诊确诊的育龄期BV患者35例和同期入院体检的健康体检女性37例,无菌拭子采集阴道中段壁分泌物,提取细菌基因组DNA,采用实时定量PCR技术（real-time quantitative PCR,qPCR）进行阴道优势细菌检测,并进行其与临床指标如阴道pH和Nugent评分相关性分析。结果 乳杆菌属细菌及其种水平的细菌如惰性乳杆菌、卷曲乳杆菌、詹氏乳杆菌等在BV患者中均显著下降,BV相关阴道致病细菌如加德纳菌属、奇异菌属、埃格特菌属、巨型球菌I型菌属、纤毛菌属和普氏菌属显著升高（P<0.05）。阴道致病菌群与阴道pH和Nugent评分呈显著正相关,而阴道卷曲乳杆菌和惰性乳杆菌与其呈负相关。结论 育龄期BV患者阴道优势菌群显著失衡,并与阴道pH和Nugent评分显著相关,提示阴道优势菌群改变参与BV发生发展。     

实时荧光定量PCR快速鉴别新型冠状病毒主要变异株北大核心CSCD

为建立一种快速鉴别严重急性呼吸综合征冠状病毒2 (severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)的5种主要变异株的Taq Man探针实时荧光定量PCR(real-time quantitative PCR, RT-qPCR)体系,基于SARS-CoV-2野生型及变异株alpha (N501Y、HV69-70del)、beta (E484K、K417N)、gamma (K417T、V1176F)、delta (L452R、T478K)和omicron (H655Y、N679K、P681H)序列设计特异性引物、探针,建立和优化一种鉴别新型冠状病毒(SARS-CoV-2) 5种主要变异株的Taq Man探针RT-qPCR方法,并进行该方法的特异性、敏感性、鉴别能力评价。该方法可准确区分出SARS-CoV-2野生型和突变型,与其他呼吸道病原体(n=21)无交叉,显示高特异性。该方法最低检测限为2×10;拷贝/mL,操作简单、快速、成本廉价,可用于监测SARS-CoV-2毒株的变异,精准指导疫情识别与防控。     

Detection of Mycobacterium immunogenum by real-time quantitative Taqman PCR

A quantitative real-time 5′-nuclease (Taqman) PCR technique was developed to specifically detect Mycobacterium immunogenum. rpoB-specific primers and Taqman probe were evaluated for detection of M. immunogenum DNA extracted from pure cultures and from industrial metal working fluids (MWFs). Specificity was confirmed and the sensitivity of detection of M. immunogenum genomic DNA was shown to be approximately 9 fg (2 cell equivalents). When tested on industrial metal working fluids from the UK and USA from which no M. immunogenum CFU were recovered, the assay detected between 3.4 × 10¹ and 1.9 × 10⁴ cell equivalents (CE) per ml, and increased the detection rate over culture to 37.5% (12 of 32 samples). This assay provides a specific, sensitive and rapid method for the detection of M. immunogenum and is applicable within industry for the early detection of this human pathogen and to the possible prevention of hypersensitivity pneumonitis (HP) in workers.