Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology

AIM: To develop a real-time PCR for detecting hepatitis B virus-(HBV) DNA based on TaqMan technology using a new MGB probe.METHODS: Plasmid containing the sequence of X gene (1414-1744 nt) was constructed as HBV-DNA standard for quantitative analysis. A TaqMan-MGB probe between primers for amplification was designed to detect PCR products. The interested sequence contained in the plasmid and in clinical specimens was quantitatively measured.RESULTS: The detection limit of the assay for HBV DNA was 1 genome equivalent per reaction. A linear standard curve was obtained between 100 and 109 DNA copies/reaction (r＞0.990). None of the negative control samples showed false-positive reactions in duplicate. HBV DNA was detected in 100% (50/50) of HBV patients with HbeAg, and in 72.0% (36/50) with HBsAg, HBeAb and HBcAb. The coefficient of variation for both intra- and inter-experimental variability demonstrated high reproducibility and accuracy.CONCLUSION: Real-time PCR based on TaqMan-MGB probe technology is an excellent method for detection of HBV DNA.

TaqMan-MGB荧光定量IC/TC-RT-PCR检测菜豆荚斑驳病毒

菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)是我国重要的检疫性有害生物,BPMV传入的风险随大豆进境数量增多而加大。本文针对菜豆荚斑驳病毒,以大豆种子提取液为材料,分别建立了TaqMan-MGB荧光定量IC-RTPCR和TaqMan-MGB荧光定量TC-RT-PCR快速检测方法。根据GenBank公布的BPMV外壳蛋白基因序列,选择其保守区域,设计1对特异性引物和1条TaqMan-MGB探针,测定了TaqMan-MGB荧光定量IC-RT-PCR和TaqMan-MGB荧光定量TC-RT-PCR方法的特异性和灵敏度,并将TaqMan-MGB荧光定量IC-RT-PCR、TaqMan-MGB荧光定量TC-RTPCR、IC-RT-PCR和TC-RT-PCR 4种检测方法的灵敏度进行比较。结果表明:所建立的两种检测方法特异性良好;TCRT-PCR的灵敏度为10-1倍病毒提取液原液;IC-RT-PCR和TaqMan-MGB荧光定量TC-RT-PCR灵敏度相当,为10-3倍病毒提取液原液;TaqMan-MGB荧光定量IC-RT-PCR灵敏度最高,为10-5倍病毒提取液原液,是IC-RT-PCR和TaqMan-MGB荧光定量TC-RT-PCR检测方法的102倍,是TC-RT-PCR检测方法的104倍;两种检测方法均能较好应用于实际大豆样品的检测。本文所建立的TaqMan-MGB荧光定量IC/TC-RT-PCR检测方法利用抗原特异性或试管非特异性捕捉病毒粒子,无需提取RNA,为进境大豆种子上BPMV的检测提供了稳定、快速、简便的技术依据,其较高的灵敏度和特异性具有较好的应用价值。

检测牛感染犬新孢子虫的TaqMan-MGB Real-time PCR方法的建立

为建立牛感染犬新孢子虫的Real-time PCR检测方法,根据犬新孢子虫Nc2基因保守序列设计特异性检测引物和TaqMan-MGB探针,建立新孢子虫病TaqMan-MGB Real-time PCR检测方法。PCR扩增产物约为150bp,与预期片段大小相符;Real-time PCR扩增表明,Ct值与梯度稀释的阳性质粒模板呈良好的线性关系;当检测牛源性弓形虫、牛环形泰勒和牛巴贝斯等虫种阳性DNA时均为阴性;经3D数字PCR判定,Real-time PCR方法的最低有效检测量为6.41拷贝/μL,灵敏性是常规PCR的1 000倍;重复性试验的组内平均变异系数为1.108%,组间为2.732%;本方法与《新孢子虫病检疫技术规范》(SN/T 3499-2013)的检测符合率为100%(46/46)。该Real-time PCR方法可用于早期诊断和日常监测家畜感染犬新孢子虫。

Establishment and evaluation based of a RIG-G gene detection system by TaqMan-MGB probe real-time PCR

Objective To establish a Taq Man-MGB fluorescent probe characterized real-time polymerase chain reaction(q PCR)method for detecting retinoic acid induced genes G(RIG-G)in human acute promyelocytic leukemia(M3).Analyze RIG-G expression levels in peripheral blood of both normal persons and M3 patients and

实时荧光定量聚合酶链反应检测血清2型猪链球菌的研究

目的基于TaqMan-MGB探针实时荧光定量聚合酶链反应(Real-time PCR)技术,建立针对血清2型猪链球菌的快速检测方法。方法针对血清2型猪链球菌的csp2 J基因序列,应用Beacon Designer 7.0,设计了引物和TaqMan-MGB探针,建立Real-time PCR检测方法;把目的片段克隆到pMD18-T载体制作标准曲线;以常见致病菌及条件致病菌验证引物探针的特异性;制备血液模拟标本评价本方法在临床标本中的应用。结果由标准曲线可知该方法可以检测到100拷贝/反应体系的目的基因,特异性检测显示只有血清2型猪链球菌有荧光信号,其他常见致病菌及条件致病菌均无荧光信号,血液模拟标本中3.9×102cfu/m l的细菌含量可被检出。结论以csp2 J为目的基因建立的血清2型猪链球菌Real-time PCR检测方法灵敏度高、特异性好、快速,可用于临床感染患者标本的初步筛查。