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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
Author(s) -
Andrade T P D,
Lightner D V
Publication year - 2009
Publication title -
journal of fish diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.819
H-Index - 85
eISSN - 1365-2761
pISSN - 0140-7775
DOI - 10.1111/j.1365-2761.2009.01072.x
Subject(s) - loop mediated isothermal amplification , reverse transcription loop mediated isothermal amplification , primer (cosmetics) , reverse transcriptase , nucleic acid , biology , rna extraction , microbiology and biotechnology , taqman , rna , reverse transcription polymerase chain reaction , dna , virology , polymerase chain reaction , chemistry , genetics , gene , messenger rna , organic chemistry
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 × 10 6 to 2.2 × 10 8 IMNV copy numbers μL −1 RNA, and it was similar with the standard RNA extraction (from 1.2 × 10 6 to 6.3 × 10 7 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.