Abstract
Here we present a visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting the gene encoding the σB major outer-capsid protein of novel duck reovirus (NDRV). A set of primers, composed of two outer primers, two inner primers and two loop primers, was designed based on the gene of interest. The LAMP reaction was conducted in a traditional laboratory water bath at 65 °C for 50 min. We compared the performance of calcein/Mn2+ and SYBR Green I dyes, as well as electrophoresis on agarose gel stained with GoldView nucleic acid dye to detect the RT-LAMP-amplified products and all assays could be employed to discriminate between positive and negative specimens in visible or UV light. Our data showed that there is no cross-reaction with other viruses and the RT-LAMP technique displayed high sensitivity for detecting NDRV with a minimal detection limit of 200 fg RNA input. This assay was more sensitive than conventional PCR in detecting NDRV both in natural and experimental infection. In conclusion, the RT-LAMP technique was remarkably sensitive, specific, rapid, simple and profitable for the identification of NDRV.
Highlights
A novel duck reovirus (NDRV) disease, called “spleen necrosis disease,” “new liver disease in Muscovy ducks” or “duck hemorrhagic-necrotic hepatitis,” was recently found among several duckling species, including shelducks, Pekins, wild mallards and Muscovy in China[1]
Optimization of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for NDRV detection
The results showed that RT-LAMP at 65 °C produced very clear and bright bands
Summary
A novel duck reovirus (NDRV) disease, called “spleen necrosis disease,” “new liver disease in Muscovy ducks” or “duck hemorrhagic-necrotic hepatitis,” was recently found among several duckling species, including shelducks, Pekins, wild mallards and Muscovy in China[1]. A reverse transcription loop mediated isothermal amplification (RT-LAMP) provides a rapid and precise detection assay for viral pathogens with high sensitivity and specificity[4]. This technique is profitable and convenient, only requires a constant temperature water bath and averts some deficiencies, including high necessity for equipment, tremendous cost, lengthened examination period and low sensitivity[5]. The potential application of RT-LAMP assay using the S3 gene of NDRV-NPO3 strain for specific diagnosis of NDRV infection with limited sensitivity and without its utilization in the detection of NDRV in naturally- and experimentally-infected ducks have been reported[16].
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