Development of quantitative multiplex DNA amplification method by loop-mediated isothermal amplification (LAMP) using Qprobe.
| Project/Area Number |
22590539
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory medicine
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| Research Institution | Fujita Health University |
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Principal Investigator |
IHIRA Masaru 藤田保健衛生大学, 医療科学部, 准教授 (10290165)
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| Co-Investigator(Kenkyū-buntansha) |
SUGIYAMA Hiroko 藤田保健衛生大学, 医学研究科, 研究員 (10387714)
ENOMOTO Yoshihiko 藤田保健衛生大学, 医学研究科, 研究員 (00387713)
YOSHIKAWA Tetsushi 藤田保健衛生大学, 医学部, 教授 (80288472)
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| Project Period (FY) |
2010 – 2012
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| Project Status |
Completed (Fiscal Year 2012)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2012: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2011: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2010: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | 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 |
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| Research Abstract |
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.
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Report
(4 results)
Research Products
(43 results)
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[Journal Article] Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts.2012
Author(s)
Ihira M, Enomoto Y, Kawamura Y, Nakai H, Sugata K, Asano Y, Tsuzuki M, Emi N,Goto T, Miyamura K, Matsumoto K, Kato K,Takahashi Y, Kojima S, Yoshikawa T.
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Journal Title
J Med Virol.
Volume: 84(9)
Pages: 1388-95
Related Report
Peer Reviewed
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[Journal Article] Analysis of rotavirus antigenemia in hematopoietic stem cell transplant recipients.2012
Author(s)
Sugata K, Taniguchi K, Yui A, Nakai H, Asano Y, Hashimoto S, Ihira M, Yagasaki H, Takahashi Y, Kojima S, Matsumoto K, Kato K, Yoshikawa T.
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Journal Title
Transpl Infect Dis.
Volume: 14(1)
Pages: 49-56
Related Report
Peer Reviewed
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