Development of quantitative multiplex DNA amplification method by loop-mediated isothermal amplification (LAMP) using Qprobe.

使用 Qpr​​obe 通过环介导等温扩增 (LAMP) 开发定量多重 DNA 扩增方法。

• 批准号: 22590539
• 负责人: IHIRA Masaru
• 金额: $ 2.91万
• 依托单位: Fujita Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2010
• 资助国家: 日本
• 起止时间: 2010 至 2012
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22590539/
• 关键词: 臨床検査医学; HHV6; Qprobe; GCV耐性; U69; VZV; SNP識別; サイクリングプローブ; Multiplex PCR; LAMP; real-time PCR法; HSV

The loop-mediate isothermal amplification method (LAMP) amplifies DNA with high specificity efficiency, and speed. However, the LAMP is insufficient for quantification of target DNA in comparison to the real-time PCR. In this study, we developed quantitative LAMP assay for HSV-1 and HSV-2 by Qprobe, which is quenched via electron transfer between the dye and guanine base at particular position. In singleplex reaction, the quantitative LAMPs amplified HSV DNA within 15min. The quantitative LAMP for HSV-1 and HSV-2 had high sensitivity and reproducible. However, it was impossible to determine an optimal condition for multiplex amplification. In addition, a new molecular method by using Qprobe was evaluated for screening of GCV resistant HHV-6B. The results of the new molecular method were consistent with those of the direct sequence. The sequence-specific DNA-RNA chimeric probe (cycling probe) is also appropriate method for the detection of SNP. The combination of LAMP and cycling probe might be good tool for quantitative analysis. As the first step, we developed quantitative assay combine real-time PCR with cycling probe to discriminate wild type and Oka-vaccine strain of VZV.

环介导等温扩增方法（LAMP）以高特异性、效率和速度扩增DNA。然而，与实时PCR相比，LAMP不足以定量靶DNA。本研究建立了一种以Q探针为探针的HSV-1和HSV-2的LAMP定量检测方法。在单重反应中，定量的LAMPs在15 min内扩增HSV DNA。HSV-1和HSV-2的LAMP定量检测灵敏度高，重复性好。然而，不可能确定多重扩增的最佳条件。此外，我们还建立了一种新的分子生物学方法--Q探针法，用于筛选HHV-6 B的抗GCV株。新的分子生物学方法的结果与直接测序的结果一致。序列特异性DNA-RNA嵌合探针（循环探针）也是检测SNP的合适方法。LAMP和循环探针的结合可能是一种很好的定量分析工具。作为第一步，我们建立了联合收割机实时荧光定量PCR与循环探针相结合的VZV野毒株和Oka疫苗株的定量检测方法。

项目成果

Host factors associated with the kinetics of EBV DNA load in patients with primary EBV infection.

与原发性 EBV 感染患者 EBV DNA 载量动力学相关的宿主因素。

• 发表时间: 2011
• 期刊: Microbiol Immunol.
• 作者: Nakai H;Kawamura Y;Sugata K;Sugiyama H;Enomoto Y;Asano Y;Ihira M;Ohashi M;Kato T;Yoshikawa T.
• 通讯作者: Yoshikawa T.

Suitable experimental condition for detection of Mycobacterium tuberculosis DNA in urine by the loop-mediated isothermal amplification (LAMP) method Scientific meeting for HIV/AIDS

环介导等温扩增 (LAMP) 法检测尿液中结核分枝杆菌 DNA 的适宜实验条件 HIV/AIDS 科学会议

• 发表时间: 2011
• 作者: M.Ihira

Different characteristics of human herpesvirus 6 encephalitis between primary infection and viral reactivation

• DOI: 10.1016/j.jcv.2011.02.002
• 发表时间: 2011-05-01
• 期刊: JOURNAL OF CLINICAL VIROLOGY
• 影响因子: 8.8
• 作者: Kawamura, Yoshiki;Sugata, Ken;Yoshikawa, Tetsushi
• 通讯作者: Yoshikawa, Tetsushi

Analysis of HHV-6 TRS copy numbers in clinical isolates: Possibility of superinfection

临床分离株中 HHV-6 TRS 拷贝数分析：重复感染的可能性

• 发表时间: 2013
• 作者: Ihira M;Sugiyama H;Enomoto Y;Higashimoto Y;Sugata K;Asano Y;Yoshikawa T.;大山浩之,番園恵理佳,石井香好,寺本隆佑,小林典裕,太田光煕;Yoshikawa T;大山浩之,綺田尚久,山口修子,小林典裕;Kato Yuri
• 通讯作者: Kato Yuri

Development of human herpesvirus3variant specific immunoblotting assay

人类疱疹病毒3变种特异性免疫印迹检测的开发

• 发表时间: 2011
• 作者: M.Ihira;岡田卓也;Y.Enomoto;Y.Higashimoto
• 通讯作者: Y.Higashimoto