Acute respiratory infections cause major public health problems which could lead to high morbidity and mortality, especially among young children, the elderly and immunocompromised people[1]. The etiological agents for acute respiratory infections include bacteria, viruses and fungi, in which viruses account for 80% of them [2]. Before the emergence of SARS-CoV-2, the focus of attention of viruses which causes the acute respiratory infections is influenza virus, because its high mutation rates allow the production of novel strains and therefore possible widespread dissemination of infection [7]. Though the incidence of influenza virus reduced during the COVID-19 period, due to governmental prevention strategies and personal healthy behaviors [6, 10], recent data indicated that the influenza activity began to increase. Angiezel Merced-Morales et al showed that the influenza activity rose in November and remained elevated until mid-June during the 2021-22 season in US[11], while an influenza surveillance weekly report from national influenza center of China showed a steady increase of influenza from April 2022 in southern China[13], suggesting more attention should be paid to influenza again.
Since many viruses may cause acute respiratory infections with similar symptoms and clinical features, the identification of etiological agents relies on laboratory testing. Currently, for the diagnosis of influenza virus, culture[8], hemagglutination-inhibition assay[14], colloidal gold assay[9], and more sensitive and specific nucleic acid amplification testing (NAAT) assays[3–5, 15, 16, 19] are applied. Previously we also developed a duplex RT-qPCR assay to type influenza A and B (data not shown), but the assay could not subtype influenza A virus into the most commonly detected H3N2 and H1N1(pdm09). Though several groups developed multiplex RT-qPCR assays for the subtyping of influenza virus A, they were not in “one pot” style, which either combined two or more separate multiplex RT-qPCR assays[16, 19], or only used H1 for H1N1(pdm09) and H3 for H3N2 (no N1 and N2 was detected which may result in false diagnosis)[3, 4]. An assay which could simultaneously subtype influenza A virus into H1, H3, N1 and N2 using one pot quadruplex RT-qPCR assay remains not yet reported. In the current study, an one pot quadruplex RT-qPCR assay was established for the simultaneously subtyping influenza A virus into H1, H3, N1 and N2. Specificity, sensitivity, repeatability and accuracy of the assay were first determined and then clinical samples were used to evaluate the efficacy of the assay for its potential clinical application.
All primers and probes used in the assay came from WHO guideline[18] with in-house modifications, the using of degenerate bases, which could address the genetic variability of influenza virus, and were listed in Table 1. A total of 443 HA and NA sequences from influenza A H1N1(pdm09) and H3N2 virus were obtained from National Center for Biotechnology Information (NCBI) GenBank database and aligned with CLUSTALX software. The conserved regions were retained and variable regions were replaced with degenerate bases. The one pot quadruplex RT-qPCR assay was then established via several rounds of optimization by adjusting concentration of the primers and probes, and amplification parameters. After optimization, 5µl of RNA template was added to 12.5µl of 2×OneStep RT-qPCR buffer, 0.25µl Pro Taq HS DNA Polymerase, 0.25µl Evo M-MLV RTase Enzyme mix (AG biotechnology, Hunan, China), 0.1µM HA and NA specific primers of H3N2, 0.05µM HA and NA specific probes of H3N2, 0.2µM HA and NA specific primers of H1N1(pdm09), 0.1µM HA and NA specific probes of H1N1(pdm09), and 6.63µl of ddH2O. Cycling parameters were 42°C 5 minutes, 95°C 30 seconds, followed by 40 cycles of 95°C 5 seconds, 60°C 30 seconds. Fluorescent signal was acquired on BioRad CFX96 Real-Time PCR System (Bio-Rad, California, United States) at the 60°C step at FAM, HEX, TexRed, and Cy5 channels for H3, N2, H1 and N1, respectively.
The sensitivity, which was expressed as the limit of detection (LOD), of the assay was determined by amplifying serially diluted in vitro transcribed H3, N2, H1 and N1 RNA templates as described previously[12]. For H1, N1, and H3, the limit of detection (LOD) can reach as low as 101 copies/reaction (Figs. 1a, 1b and 1c), while for N2, the LOD was 102 copies/reaction (Fig. 1d). Yang et al developed four duplex RT-qPCR assays for the typing and subtyping of influenza virus and found that the LODs of the H1, N1, H3 and N2 (detected by different duplex RT-qPCR assays) were 104, 102, 101, 103 copies/reaction [19], respectively, which was 1000 (for H1) or 10-fold (for N1 and N2) less sensitive than our one pot quadruplex RT-qPCR assay. The N1 and N2 subtype duplex RT-qPCR assay and H1, H3, H5, and NS gene quadruplex RT-qPCR assay developed by Fang et al showed a LOD of 102, 101, 102, 102 copies/µl for H1, N1, H3 and N2, which was also 10-fold (for H1 and H3) less sensitive than our assay[16]. The LODs of assays developed by He et al [5] and Ruiz-Carrascoso et al [15] showed similar results and was comparable to our assay. However, all these studies could not detect H1, N1, H3 and N2 in one single assay. These results showed that the one pot quadruplex RT-qPCR assay developed in the current study was not only sensitive but also convenient since it could subtype influenza virus into H1, N1, H3 and N2 in one pot instead of several individual assays as reported previously.
The specificity of the assay was tested by running the assay for the detection of oral swab samples from patients with influenza A H1N1(pdm09) virus, influenza A H3N2 virus, influenza B virus, along with other respiratory pathogens, including SARS-CoV-2, adenovirus, respiratory syncytial virus, human rhinovirus, streptococcus hemolytic, streptococcus pneumoniae, and haemophilus influenza infection. Only influenza A H1N1(pdm09) and H3N2 virus could be detected at FAM, HEX, TexRed, Cy5 channels for H3, N2, H1 and N1, respectively. When other respiratory pathogens were used as targets, no amplification curve could be obtained. These results indicated that the one pot quadruplex RT-qPCR assay was highly specific for the detection and subtyping influenza A H1N1(pdm09) and H3N2 virus.
1×103 copies of RNA of H3, N2, H1 and N1 was also tested 8 times to evaluate the repeatability of the assay. The cycling threshold value of the assay was recorded to determine the coefficient of variation (CV) of the assay. A CV value < 5% was regarded as good repeatability as demonstrated previously [17]. As shown in Fig. 2, all amplification curves showed good uniformity. As calculated, the CV of the assay was 1.06%, 1.33%, 1.63% and 0.64% for H1, N1, H3, and N2, respectively, far less than 5%, indicating low variability (good repeatability) of the assay.
Blinded labeled influenza A H1N1(pdm09) virus, influenza A H3N2 virus, influenza B virus, SARS-CoV-2, adenovirus, respiratory syncytial virus, human rhinovirus, streptococcus hemolytic, streptococcus pneumoniae, and haemophilus influenza samples were also prepared and tested in parallel by the assay and commercial RT-qPCR assays (four individual RT-PCR assays targeting H3, N2, H1 and N1, manufactured by Shanghai Zhijiang Bio-Tech, Shanghai, China) to determine the accuracy of the assay. Results showed that the one pot quadruplex RT-qPCR assay exhibited a 100% coincidence rate with commercial RT-qPCR assays, which indicated that the assay could be used as an potential and efficient tool for the detection and subtyping influenza A H1N1(pdm09) and H3N2 virus clinically.
After the establishment, the one pot quadruplex RT-qPCR assay was used to test clinical oral swab samples with the aim to evaluate the clinical performance of the assay. For this purpose, a total of 376 oral swab samples were collected from entry personnel during January to June, 2022. This was conducted in an anonymous way in line with guidelines set by the local ethical review committee. Informed consent was obtained from each study participant. These samples were tested using the one pot quadruplex RT-qPCR assay and commercial RT-PCR assays. 137 out of 376 samples (137 positive for subtype H3N2, 36.44%) were confirmed positive both by the one pot quadruplex RT-qPCR assay and commercial RT-qPCR assays. No influenza A H1N1(pdm09) virus was detected. Furthermore, 17 out of 376 samples (4.52%) were confirmed positive for influenza B by in house duplex RT-qPCR assay as mentioned previously. Majority of the viruses detected among those samples were influenza A H3N2 virus, which was in agreement with the national influenza surveillance report from China[13], suggesting the predominant influenza virus during this wave was influenza A H3N2 virus. A recent study showed that most of the influenza A H3N2 viruses belonged to the 3C.2a1b.2a.2 subclade and were antigenically distinct from the vaccine components for the 2021–22 Northern Hemisphere influenza vaccines[11]. Whether our influenza A H3N2 viruses were also antigenically different from the vaccine components needed further investigation.
In conclusion, a one pot quadruplex RT-qPCR assay was established in the current study. The assay would be highly suitable for the sensitive and specific detection and subtyping influenza virus into H1, N1, H3 and N2 in an one-pot, quadruplex format.