Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of newly emerged H5N6 influenza viruses
- PDF / 947,214 Bytes
- 7 Pages / 595.276 x 790.866 pts Page_size
- 114 Downloads / 212 Views
RESEARCH
Open Access
Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of newly emerged H5N6 influenza viruses Lin Liu, Ying Zhang, Pengfei Cui, Congcong Wang, Xianying Zeng, Guohua Deng and Xiurong Wang*
Abstract Background: In 2017–2018, a new highly pathogenic H5N6 avian influenza virus (AIV) variant appeared in poultry and wild birds in Asian and European countries and caused multiple outbreaks. These variant strains are different from the H5N6 virus associated with human infection in previous years, and their genetic taxonomic status and antigenicity have changed. Therefore, revision of the primers and probes of fluorescent RT-PCR is important to detect the new H5N6 subtype AIV in poultry and reduce the risk of an epidemic in birds or humans. Methods: In this study, the primers and probes including three groups of HA and four groups of NA for H5N6 influenza virus were evaluated. Then a set of ideal primer and probes were selected to further optimize the reaction system and established a method of double rRT-PCR assay. The specificity of this method was determined by using H1~H16 subtype AIV. Results: The results showed that fluorescence signals were obtained for H5 virus in FAM channel and N6 virus in VIC channel, and no fluorescent signal was observed in other subtypes of avian influenza viruses. The detection limit of this assay was 69 copies for H5 and 83 copies for N6 gene. And, the variability tests of intra- and inter-assay showed excellent reproducibility. Moreover, this assay showed 100% agreement with virus isolation method in detecting samples from poultry. Conclusion: The duplex rRT-PCR assay presented here has high specificity, sensitivity and reproducibility, and can be used for laboratory surveillance and rapid diagnosis of newly emerged H5N6 subtype avian influenza viruses. Keywords: Duplex rRT-PCR, H5N6 virus, Avian influenza virus
Background Avian influenza viruses (AIVs) belong to the Orthomyxoviridae family with a natural reservoir almost entirely in birds [1]. The virus particle contains a genome that includes eight separate negative-stranded RNA segments and majorly adopts an elliptical shape [2]. The two large proteins found on the surface of the viral envelope are hemagglutinin (HA) and neuraminidase (NA) [3]. Based on the antigenicity of these two proteins, AIVs are categorized into different subtypes [4]. Currently, 16 different HA and 9 different NA subtypes of AIVs have been identified [5]. * Correspondence: [email protected] National Avian Influenza Reference Laboratory, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China
H5 subtype AIVs can be classified into two groups based on their pathogenicity on chickens: high pathogenicity and low pathogenicity groups [6]. Highly pathogenic H5 subtype AIVs have infected wild birds, and continued to cause outbreaks in poultry [7]. Besides, these viruses have also caused sporadic infections in humans, thus po
Data Loading...
