۱٫ Introduction

۲٫ Material and methods

۳٫ Results

۴٫ Discussion

۵٫ Conclusions

Acknowledgements

References

Abstract

Marek’s disease (MD) is one of the most devastating diseases of poultry. It’s caused by the highly infectious alphaherpesvirus MD virus serotype 1 (MDV-1). In this study, a rapid and easy-to-use assay based on recombinase polymerase amplification (RPA) was developed for MDV detection. Primer-probe sets targeting the highly conserved region of Meq gene were designed and applied to the RPA assay. The assay was carried out on a real-time thermostatic fluorescence detector at 39 °C for 20 min. As revealed by the results, no cross-reactions were found with the Newcastle disease virus (NDV), chicken infectious anemia virus (CAV), infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avain influenza virus (AIV), avian leucosis virus (ALV), avian reovirus (ARV), Marek’s disease virus serotype 2 (MDV-2) and turkey herpes virus (HVT), indicating appropriate specificity of the assay. Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 102 copies/μL. To further evaluate the clinical performance, 94 clinical samples were subjected to the RPA assay and 28 samples were tested MDV positive, suggesting that the real-time RPA assay was sufficient enough for clinical sample detection. Thus, a highly specific and sensitive real-time RPA assay was established and validated as a candidate for MDV diagnosis. Additionally, the portability of real-time RPA assay makes it suitable to be potentially applied in clinical diagnosis in the field, especially in resource-limited settings.

Introduction

Marek’s Disease Virus (MDV) is the causative agent of Marek’s disease (MD) in chickens [1]. It is a highly contagious oncogenic herpesvirus of poultry that can infect chickens, turkeys and pheasant. The virus is shed from feather follicles of infected chicken or birds and contaminates the living environment. Chickens will be infected by inhalation of contaminated air [2,3]. The prevalence of MD in all poultryrearing countries causes huge economic losses every year [4,5]. Isolates of MDV can be classified into three serotypes, only serotype 1 (MDV1) or Gallid herpesvirus 2 (GaHV-2) is pathogenic and oncogenic. The severity of MD varies depending on virus stains, host genotypes and the vaccination status of the infected hosts [6]. Despite the wide use of vaccines, the economic burden of the disease will substantially increase. Therefore, timely and accurate detection of MDV in poultry farms is extremely important.

Conventional methods such as virus isolation and serum neutralization tests are labor-intensive and time-consuming [7]. To date, real-time PCR, GeXP-multiplex PCR assays and xTAG assay have been developed for MDV detection [8–۱۰]. These methods are specific, sensitive and even enable high-throughput detection, which make them perfect choices for laboratory diagnosis. However, the need of expensive equipment such as Luminex-200 reader or GeXP analyser and well-trained technicians limits their applications in poultry farms [11,12].