Development of a method for the detection of infectious myonecrosis virus by reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow hybrid assay

T. P.D. Andrade, D. V. Lightner

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

We report the development of a reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow method (RT-LAMP-NALF) for detection of infectious myonecrosis virus (IMNV). The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The sensitivity of RT-LAMP (using two and three primer pairs) and nested RT-LAMP (using three primer pairs) was compared by real-time reverse-transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. The detection of RT-LAMP (three primer pairs) products was accomplished by using a NALF-test strip. The RT-LAMP-NALF showed equivalent sensitivity to RT-LAMP (using three primer pairs), and it was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. On the other hand, the RT-LAMP-NALF was 10 and 100 times less sensitive than nested RT-PCR and real-time RT-PCR, respectively. The simplified RNA extraction method ranged from 4.4 × 106 to 2.2 × 108 IMNV copy numbers μL-1 RNA, and it was similar with the standard RNA extraction (from 1.2 × 106 to 6.3 × 107 IMNV copy numbers μL-1 RNA). These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.

Original languageEnglish (US)
Pages (from-to)911-924
Number of pages14
JournalJournal of Fish Diseases
Volume32
Issue number11
DOIs
StatePublished - Nov 1 2009

Keywords

  • Infectious myonecrosis virus
  • Lateral flow assay
  • Litopenaeus vannamei
  • Nucleic acid lateral flow method
  • Reverse-transcription loop-mediated isothermal amplification

ASJC Scopus subject areas

  • Aquatic Science
  • veterinary (miscalleneous)

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