gms | German Medical Science

International Conference on SARS - one year after the (first) outbreak

08. - 11.05.2004, Lübeck

RT-LAMP method provides a simple, rapid and specific detection system for SARS-CoV RNA

Talk

  • T. Notomi - National Institute of Infectious Diseases, Japan; Eiken Chemical co., Ltd
  • corresponding author presenting/speaker F. Taguchi - National Institute of Infectious Diseases, Japan
  • H. Kanda - National Institute of Infectious Diseases, Japan; Eiken Chemical co., Ltd
  • H. Minekawa - National Institute of Infectious Diseases, Japan; Eiken Chemical co., Ltd
  • S. Itamura - National Institute of Infectious Diseases, Japan
  • T. Odagiri - National Institute of Infectious Diseases, Japan
  • M. Tashiro - National Institute of Infectious Diseases, Japan

International Conference on SARS - one year after the (first) outbreak. Lübeck, 08.-11.05.2004. Düsseldorf, Köln: German Medical Science; 2004. Doc04sars8.09

Die elektronische Version dieses Artikels ist vollständig und ist verfügbar unter: http://www.egms.de/de/meetings/sars2004/04sars045.shtml

Veröffentlicht: 26. Mai 2004

© 2004 Notomi et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

LAMP is a novel nucleic acid amplification method that amplifies DNA with simplicity, rapidity and high specificity under isothermal conditions. The amplified products can reach as much as 10ug/tube, so that a simple detection can be attained. Based on the features of LAMP method, we have developed a detection system for coronavirus that caused the new emerging infectious disease SARS.

LAMP primers for SARS-CoV RNA were designed from the Replicase 1b region. At constant temperature of 63 C, RT-LAMP reaction was conducted in 25ul of the reaction mixture, which contains AMV reverse transcriptase, Bst DNA polymerase, dNTPs and the primers. The formation of tremendous amount of by-product pyrophosphate can be used as an indicator for the detection of the amplified product, by which the real-time detection can be achieved by monitoring the precipitate of magnesium pyrophosphate, or by visually observing the increasing fluorescence intensity as the fluorescent chelating reagent responds to the change of metal ion concentration in the mixture due to the formation of magnesium pyrophosphate. The reaction was carried out using the real-time turbidimeter LA200 (TERAMECS, Japan). RNA was isolated from the sera or plasmas using QIAamp viral RNA mini kit (Qiagen, Germany), and a part of the elution was 3-5 times concentrated by isopropanol.

Using RT-LAMP method, the target regions in either in vitro transcript or inactivated genomic RNA could be detected within 45 minutes with the sensitivity being 10 copies/test. No amplification was observed when the reactions were conducted on the extraction of the normal sera and plasmas from 47 samples. In addition, no cross-reactivity with detection system was recognized against other coronaviruses, influenza virus and RS virus. The simple RNA enrichment method could increase the efficiency of detection for SARS-CoV RNA whose concentration is usually low in serum. Moreover, visual detection using fluorescence could also be conducted.

Conclusively, RT-LAMP method can provide a highly specific, rapid and simple detection system for SARS-CoV.