Real-time fluorescence loop-mediated isothermal amplification assay for direct detection of egg drop syndrome virus

Makay Zheney,Lingna Zhao,Gang Li,Gulmira Kassenova,Wei Liu,Lin Liang,Zhambul Kaziyev

doi:10.1186/s12917-018-1364-9

Abstract

BackgroundEgg drop syndrome (EDS), caused by the adenovirus “egg drop syndrome virus” (EDSV) causes severe economic losses through reduced egg production in breeder and layer flocks. The diagnosis of EDSV has been done by molecular tools since its complete genome sequence was identified. In order to enhance the capabilities of the real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay, we aimed to apply the method for direct detection of the EDSV without viral DNA extraction. In order to detect the presence of the EDSV DNA, three pairs of primers were designed, from the conserved region of fiber gene of the EDSV.ResultsFor our assay, test and control samples were directly used in the reaction mixture in 10-fold serial dilution. The target DNA was amplified at 65 °C, which yield positive results in a relatively short period of 40–45 min. The method reported in this study is highly sensitive as compared to polymerase chain reaction (PCR) and showed no sign of cross-reactivity or false positive results. The RealAmp accomplished specific identification of EDSV among a variety of poultry disease viruses.ConclusionsThe direct RealAmp can be used to detect the presence of EDSV. As our result showed, the RealAmp method could be suitable for the direct detection of other DNA viruses.

Highlights

Egg drop syndrome (EDS), caused by the adenovirus “egg drop syndrome virus” (EDSV) causes severe economic losses through reduced egg production in breeder and layer flocks
realtime fluorescence loop-mediated isothermal amplification (RealAmp) of EDSV DNA In this assay, the EDSV DNA was successfully amplified from diluted viral DNA samples extracted from infected allantoic fluid and cell culture supernatants within 40 min (Fig. 2a and b)
Direct RealAmp assay By using infected allantoic fluid and cell culture supernatants, as well as undiluted samples directly in the assay, we successfully amplified EDSV DNA

Summary

Introduction

Egg drop syndrome (EDS), caused by the adenovirus “egg drop syndrome virus” (EDSV) causes severe economic losses through reduced egg production in breeder and layer flocks. In order to enhance the capabilities of the realtime fluorescence loop-mediated isothermal amplification (RealAmp) assay, we aimed to apply the method for direct detection of the EDSV without viral DNA extraction. Loop-mediated isothermal amplification (LAMP) is a method that can amplify DNA under isothermal conditions. It was first developed by the Japanese researchers, the LAMP employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA [12]. LAMP has some advantages in comparison with PCR methods, including improved sensitivity and specificity, as well as time efficiency [13]

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Conclusion