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

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.