An emerging novel duck reovirus (NDRV) disease, called Duck Spleen Necrosis Disease, was recently found in China and the pathogen, distinct from the MDRV isolates previous, was identified to be a Orthoreovirus [1, 3]. Importantly, Wang et al. recently report that the emerging NDRV XT18 has extensive tissue tropism and could cause severe damage to the immune organs [3, 8]. Furthermore, the NDRV co-infection with other pathogens can result in serious duck diseases [4]. At present, with the frequent trade of agricultural products between countries or regions, the possibility of virus transmission is greatly increased. So, it is essential to develop molecular diagnosis techniques for the purpose of correct and rapid detection of viral pathogens in order to prevent further disease transmission or outbreaks.
Multiple diagnostic techniques, including RT-PCR, RT-LAMP and several serological assays, have been used to detect viruses in various samples [11, 13, 14]. Although test results obtained through RT-PCR are reliable, this technique is too costly and time-consuming for companies or persons who have limited laboratory equipment, including a thermal cycler, and can only conduct mostly field surveys [26]. RPA is a rapid and simple isothermal gene amplification method and does not require thermal cycler and high temperatures that can overcome the shortcomings of conventional PCR-based methods. At 37 ℃, the reaction can be completed within 20 minutes with only a pair of primers and a simple device such as water bath and heating block. Rapid and specific detection of NDRV using the RT-LAMP procedure has also been reported for ducklings [14, 15]. But RT-LAMP method requires high temperature (60–65 ℃), six primers and more difficult downstream analysis, such as cloning and direct sequencing, limitations that are overcome by the RT-RPA procedure. In our study, we first developed a new method for rapid detection of NDRV based on an isothermal gene amplification, with high sensitivity and specificity.
Currently, all kinds of modified RPA assay have been applied to the diseases diagnosis in the field of human and veterinary medicine, as well as the detection of pathogenic bacteria in the food safety and agriculture respectively [29]. The RT-RPA assays has some advantages over the ELISA, conventional RT-PCR and RT-LAMP currently used for detection NDRV due to its short reaction time and only require a single constant temperature (33 to 43 ℃). The RT-RPA assays was enough to produce sufficient amount of NDRV amplicon at 37 ℃ incubated for 3 min for rapid detection of the virus. On the contrary, conventional RT-PCR requires more than 95 min to complete before gel electrophoresis [11] and 60–65 min was required for RT-LAMP [14]; and two days were require for ELISA detection [13]. In addition, the high specificity and amplification of the RT-RPA method also allows for the easy and rapid visualization of the amplified products without the need for gel electrophoresis, thus making it a very simple and rapid diagnostic tool.
Compared with the conventional RT-PCR, RT-RPA assays has higher sensitivity and specificity in detecting NDRV. This method has high specificity and no cross-reactivity was detected with other waterfowl-origin viruses. Furthermore, the detection limit of RT-RPA was 3.48 × 10− 6 ng/µl with the standard plasmid DNA, which is a 10-fold higher sensitivity rate than that of the conventional RT-PCR previously reported. RT-LAMP and RT-PCR are both reliable methods for detecting NDRV with high sensitivity and specificity. However, aerosol pollution and false positives are prone to occur in RT-LAMP reactions [30]. The RPA method developed in our study did not produce this pollution and/or false positives, possibly since its reagents used are provided in a lyophilized pellet [31]. Finally, we evaluated the newly established RT-RPA method in this study by using field and experimental infected samples, and found that the test results were highly consistent those with the conventional RT-PCR and qRT-PCR assays.