Development and evaluation of a TaqMan MGB RT-PCR assay for detection of H5 and N8 subtype influenza virus

Background Highly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed. Methods In the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay. Results The results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction. Conclusions The developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.

]. An increasing number of reports indicate that HPAI H5N8 viruses continuously cause deaths in wild migratory birds and birds in live poultry markets [18][19][20].
For more effective prevention and control of H5N8 infection, the development of a rapid, sensitive and specific diagnostic assay is critical. Currently, viral culture is the most traditional method for influenza diagnosis, and is considered the gold standard. However, it is timeconsuming and complicated, and requires a laboratory with bio-safety level 3 practices [21]. Reversetranscription PCR (RT-PCR) is the most well-established molecular detection technology currently available to detect and/or type influenza viruses [22]. Real-time RT-PCR (RRT-PCR), developed from RT-PCR, can monitor the progress of reactions by detecting the fluorescence signal in real time, resulting in higher sensitivity, specificity and simplicity [23]. In the present study, we developed a TaqMan minor groove binder (MGB) RRT-PCR assay to detect H5N8 subtype avian influenza viruses (AIVs) rapidly and specifically.

Method
Three pairs each of specific primers and corresponding probes targeting H5 hemagglutinin (HA) and N8 neuraminidase (NA) genes were designed based on the nucleotide sequences of H5-HA (H5N1, H5N2, H5N6 and H5N8) (2.3.2 and 2.3.4.4) and N8-NA (H2N8, H3N8, H5N8, H6N8 and H10N8) genes from 1998 to 2018, obtained from the GenBank database, using Primer Express software as described previously [24]. Finally, two optimal sets of primers and probes for H5-HA and N8-NA (Table 1 and Fig. S1) were chosen after numerous comparison experiments as described previously [25]. A total of 34 strains of AIVs (Table 2) were selected to test the performance of the assay. Newcastle disease virus (NDV), infectious bronchitis (IBV) and infectious bursal disease virus (IBDV) were also used to assess specificity. And a reference real-time RT-PCR was performed using an Influenza A Virus Real Time RT-PCR Kit (Liferiver, Shanghai, China) according to the manufacturer's instructions [26].
The sensitivity of the RRT-PCR assay was determined for each reaction using 10-fold serial dilutions of H5 and N8 plasmids, with DNA ranging from 1 to 10 9 copies per reaction [28]. To evaluate the clinical sensitivity and specificity of the RRT-PCR assay, sixweek-old female BALB/c mice (n = 24) were Note: Primers and probes were targeted to the conserved regions of the H5-HA and N8-NA genes, and the H5 gene-specific probe was labelled with FAM at the 5′ end, while the N8 gene-specific probe was labelled with HEX to allow specific detection of H5N8 AIVs in a single reaction. The specific primers and probes were designed based on the nucleotide sequences of 781 H5N8-HA genes (including high pathogenic and low pathogenic H5N8), obtained from the GenBank database, using Primer Express software. By in silico analysis of published H5N8 sequence data, the primers and probes of H5 anesthetized by isoflurane and inoculated intranasally with H5N8 virus in 0.05 mL phosphate buffered saline. Respiratory specimens and cloacal swab samples were harvested from mice at 3 days post-inoculation, and the mice were sacrificed with 5% isoflurane.

Results
In this study, DNA plasmids were used for analytical sensitivity testing instead of RNA run-off transcripts as described previously [28]. The detection limit of the assay was 10 copies per reaction for both H5 and N8 genes. Standard curves for the two plasmids were generated by plotting their cycle threshold (Ct) values against DNA copy number, and both followed a linear correlation between 10 and 10 9 copies of target DNA in each multiplex detection reaction (Fig. 1). Linear correlations of the standard curves of H5 and N8 were y = − 3.407x + 40.688 (efficiency = 96.6%, R 2 = 0.991), and y = − 3.325x + 40.016 (efficiency = 99.9%, R 2 = 0.991), respectively. The diagnosis specificity of the assay was evaluated using the viruses listed in Table 2. The results showed that only H5 and N8 AIVs yielded a positive signal, while all other AIV subtypes and other viruses were negative.
Regarding reproducibility, inter-assays and intraassays were analysed using different concentrations of plasmids as described previously [29]. The results of intra-assays (Table 3) and inter-assays (Table 4) revealed that the coefficients of variation (CV%) were all < 2%, suggesting our RRT-PCR method is highly reproducible [30].
Respiratory specimens and cloacal swab samples (n = 24) from mice infected with H5N8 [27] were collected and tested to evaluate the clinical sensitivity and specificity of the RRT-PCR assay. Concurrently, samples were   China), and the results were used as a reference as described previously [24,26]. Positive signals were obtained for all H5 and N8 samples, and the results were consistent with those obtained with the Influenza A Virus Real Time RT-PCR Kit (Fig. 2).
Additionally, a total of 148 cloacal swabs were collected from poultry in Zhejiang from 2013 to 2018 [31][32][33] and tested using both the RRT-PCR assay and virus isolation. The results of the RRT-PCR assay showed that there were 12 positive samples of H5N8 subtype AIVs, six positive samples of H5Nx subtype, and eight positive samples of HxN8 subtype, consistent with the results of virus isolation (Table 5).

Discussion
Increasing evidence suggests that many subtypes of AIVs, such as H7N9, H10N8, H6N1, H9N2 and H7N7, are not only pathogenic for poultry, but they can also infect humans, and even cause death [34][35][36]. Historically, H5N1 and H7N9 AIVs have caused great economic losses and numerous deaths in humans [37,38]. H5N8 HPAI has caused multiple disease outbreaks in poultry and wild birds, and has the potential to be transmitted from birds to humans. In view of the global threat posed by the H5N8 virus, an appropriate technology for timely detection and surveillance of this virus is required. A multiplex RRT-PCR assay for detecting H5N8 has been developed previously with a detection limit of 99.9 copies per reaction for the H5 gene and 15.9 copies per reaction for the N8 gene [39]. A riems influenza a typing array (RITA) was developed by duplex TaqMan reactions for detection and identification of 14 HA and 9 NA subtypes of AIVs, including H5 and N8 subtype AIVs. But no H5N8 AIVs were included in this study to . In silico mismatches do not necessarily translate into failure of detection in the wet assay. The capacity of the RRT-PCR developed in this study to cover the above strains should be further verified. In the present work, an efficient RRT-PCR assay was developed with a detection limit of 10 copies per reaction for both H5 and N8 genes via careful design and optimisation of primers and probes. Additionally, this assay performed well in the analysis of clinical samples.

Conclusions
These results indicate that the duplex assay designed in this study is sufficiently sensitive and specific to be used for the detection of the H5N8 virus.
Additional file 1: Table S1. The optimal concentrations of H5 primers and probe. a. The most optimal concentrations of H5 primers and probe.
Additional file 2: Table S2. The optimal concentrations of N8 primers and probe. a. The most optimal concentrations of N8 primers and probe.
Additional file 3: Figure S1. Phylogenetic analysis (A and B) and sequence alignments (C and D) of the H5 and N8 genes of H5N8 influenza viruses. The tree was created by the maximum likelihood method and bootstrapped with 1000 replicates using the MEGA6 software version 6.0. The scale bar represents the distance unit between sequence pairs.