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, Donald V Lightner

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11 Citations (Scopus)

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 2009

Fingerprint

nucleic acid
Nucleic Acids
nucleic acids
Reverse Transcription
amplification
virus
assay
Viruses
viruses
assays
polymerase chain reaction
RNA
reverse transcriptase polymerase chain reaction
methodology
Polymerase Chain Reaction
quantitative polymerase chain reaction
detection
method
reverse transcription loop-mediated isothermal amplification
extraction method

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)

Cite this

@article{36b62bfd8b3546a39a7e2be418ad4485,
title = "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",
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.",
keywords = "Infectious myonecrosis virus, Lateral flow assay, Litopenaeus vannamei, Nucleic acid lateral flow method, Reverse-transcription loop-mediated isothermal amplification",
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T1 - 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

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N2 - 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.

AB - 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.

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