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

Zheney et al. BMC Veterinary Research (2018) 14:49 DOI 10.1186/s12917-018-1364-9 METHODOLOGY ARTICLE Open Access Real-time fluorescence loop-mediated isothermal amplification assay for direct detection of egg drop syndrome virus Makay Zheney1,2, Zhambul Kaziyev2, Gulmira Kassenova2, Lingna Zhao1, Wei Liu1, Lin Liang1 and Gang Li1* Abstract Background: 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. 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 realtime 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. Results: For 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. Conclusions: The 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. Keywords: Egg drop syndrome virus, Real-time fluorescence loop-mediated isothermal amplification, Sensitivity, Specificity Background Egg drop syndrome is a viral disease, caused by the egg drop syndrome virus (EDSV), officially called duck adenovirus 1 (DAdV-1), belonging to species Duck adenovirus A, genus Atadenovirus, family Adenoviridae. EDSV was first reported in 1976, it has also been known as adenovirus 127 and egg-drop-syndrome-76 (EDS-76) virus [1]. EDS is characterized by the production of softshelled, thin shelled, shell-less, and discolored eggs in otherwise healthy chickens [2]. The natural hosts of the EDSV are ducks and geese, however, the virus can also infect chickens, resulting in major economic losses on egg production [3, 4]. EDSV was involved in severe respiratory disease in 1-day-old goslings where the presence of EDSV DNA was found in different organs of the * Correspondence: 1 State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China Full list of author information is available at the end of the article naturally and experimentally infected goslings [5]. Severe acute respiratory symptoms with coughing, dyspnea, and gasping were reported in 9-day-old Pekin ducklings in 2013 [6]. For diagnosis of EDSV, five serological methods have been used and tested [7]. In the recent years, several PCR studies have been published, for diagnosis of all avian adenoviruses that are of relevance for poultry production [8–10]. Molecular amplification methods were commonly used to diagnose EDSV infection [11]. 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]. Later, LAMP was supplemented by using additional primers, termed loop primers which prime strand displacement DNA synthesis. Moreover, LAMP has some advantages in comparison with PCR methods, including improved sensitivity and specificity, as well as time efficiency [13]. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zheney et al. BMC Veterinary Research (2018) 14:49 Since LAMP was published, a range of LAMP methods have been developed. The RealAmp is one of them, which attempted to improve the method for diagnosis by using a simple and portable device capable of performing both the amplification and detection by fluorescence in one platform [14]. Currently, the LAMP assays are utilized to detect bacterial and viral pathogens including Mycobacterium tuberculosis, Acinetobacter baumannii, avian influenza virus, Middle East respiratory syndrome coronavirus and hemorrhagic enteritis virus [15–19]. Various LAMP procedures have been successfully employed for DNA amplification using DNA templates extracted from the samples. The purpose of this study was to evaluate the usability of the RealAmp method for a rapid detection of the EDSV in a diverse range of samples without a prior need for nucleic acid extraction. Therefore, we infected both duck embryos and duck fibroblast cell culture with EDSV, then the viral samples were collected and employed to the assay directly by serial dilutions. Methods Chemicals and reagents Enzymes including Bst2.0 DNA polymerase (8000 U/ml) and BsrGI-HF (20,000 U/ml) were obtained from New England Bio labs (NEB, USA). Primers for RealAmp and PCR (oligonucleotides) were obtained from Huada (Beijing, China) and suspended in deionized water with appropriate concentrations and stored at − 20 °C. The concentrations of each DNA suspension used in this study were measured by NanoVue Plus spectrophotometer (GE Healthcare, USA). Description of the equipment The fluorescence reader ESE-Quant Tube Scanner used for this study was developed by a commercial manufacturer (ESE Gmbh, Stockach, Germany). It has an eight tube holder heating block with adjustable temperature settings and spectral devices to detect amplified product using fluorescence spectra. This equipment is easy to handle, and completely portable. The results can be seen in a small monitor or on a computer screen connected to the equipment. Page 2 of 8 China. The viruses were kept in tissue culture supernatant in the laboratory at − 80 °C. Inoculation of embryonated duck eggs with the EDSV For virus propagation 9-day-old embryonated duck eggs were obtained from Beijing Dayinghongguang Duck Farm, and incubated for 2 days in an egg incubator. EDSV (EDS-NE4) used in this experiment was isolated in 1992 [20], and kept in the Animal Disease Control Laboratory of Institute of Animal Sc (...truncated)