Detection of influenza A virus by MultiCode based real-time RT-PCR

Abstract

Influenza virus belongs to the Orthomyxoviridae virus family, which is enveloped and pleomorphic with size ranging from 80-120nm. Influenza virus has a negative-sense, single-stranded, segmented RNA genome. Influenza A virus (IAV) is the most significant and widespread virus of the group since it can infect a broad range of avian and mammalian species. Also, due to high mutation rates and frequent genetic reassortment of influenza A virus, antigenic drift and antigenic shift often occur. In some rare cases, an antigenic shift may cause large global or regional pandemic outbreaks. There are several methods to detect influenza A virus in the laboratory, which includes, viral culture, direct immunofluorescence assay (DIF), rapid antigen detection kit and reverse transcription-PCR (RT-PCR). Nowadays, RT-PCR is the most common method to detect influenza A viruses, which is more sensitive and specific comparing with other options. In addition, real-time RT-PCR has short turnaround time (TAT), and it can provide quantification of viral load and identification of different subtypes of influenza A virus. The aim of this study was to evaluate a probe-free based, MultiCode®-RTx assay towards the detection of M gene of influenza A virus, and the feasibility to differentiate different subtypes of influenza A virus by the M gene melting temperature (Tm). Total nucleic acids of 111 clinical specimens and six culture isolates of pH1N1pdm09 (A/415742/09/H1N1), H2N2 (A/Asia/57/3), H3N2 (A/4851970/14), H5N1 (A/Vietnam/3028/04), H7N9 (A/Anhui/1/2013) and H9N2 (A/HK/1073/99) were included in this study. First, the performance of the MultiCode®-RTx assay for detection of influenza A infection was compared with the reference test, Queen Mary Hospital in-house real-time RT-PCR assay. The overall concordance of both assays was analyzed by the Cohen’s kappa value. The limit of detection (LOD) according to HA subtypes was determined by using the culture isolates. The sensitivity of MultiCode®-RTx M gene assay for overall influenza A, H1 and H3 subtype from clinical specimens was 88.9%, 93.3% and 87.5% respectively. The specificity was 100% and the overall PPV and NPV was 100% and 87.3%. The Cohen’s kappa value was 0.874 which is considered very good agreement between MultiCode®-RTx assay and in-house RT-PCR. The LOD of M gene with H1, H2, H3, H5, H7 and H9 subtypes was 67.5, 575, 67.5, 6.75, 10 and 550 TCID50 respectively. The Tm of H1N1 (clinical specimens), H3N2 (clinical specimens), H1N1 (culture isolate), H2N2 (culture isolate), H3N2 (culture isolate), H5N1 (culture isolate), H7N9 (culture isolate) and H9N2 (culture isolate) was 83.2 °C (sd ± = 0.14), 82.9 °C (sd ± = 0.14), 83.3 °C (sd ± = 0.07), 84.3 °C (sd ± = 0.04), 82.8 °C (sd ± = 0.04), 84.0 °C (sd ± = 0.04), 83.2 °C (sd ± = 0.04) and 83.9 °C (sd ± = 0.11) respectively. The Tm of different HA subtypes found in this study could be categorized into two groups; one was the Tm around 83°C (sd ± = 0.09) which included H1, H3 and H7 HA subtypes. Another was the Tm around 84°C (sd ± = 0.06) which included H2, H5 and H9 HA subtypes. Although only two Tm groups could be categorized, this 1°C difference was enough to differentiate these two groups of HA subtypes and facilitate the clinical diagnosis. In conclusion, in-house MultiCode®-RTx assay for M gene was showed highly specific, less sensitive than in-house M gene assay but comparable with some of the commercial multiplex PCR kits. It is suitable for use in clinical diagnostic laboratories for diagnosis of influenza infection. It can also provide additional information to distinguish H7N9 and H5N1/H9N2 influenza infection which are highly pathogenic avian influenza (HPAI) viruses.