Development of a Diagnostic System for Novel Influenza A(H7N9) Virus Using a Real-Time RT-PCR Assay in Japan

Abstract

SUMMARY:The first human cases of infection with avian influenza A(H7N9) virus were reported in March 2013 in China. The number of confirmed cases continues to increase, although almost all the cases are limited to China. In this study, a one-step real-time RT-PCR assay was developed for detecting the novel A(H7N9) virus. This assay was shown to have high specificity, good linearity, and high sensitivity to a broad range of Eurasian H7 viruses. The assay is useful both for diagnostic purposes in cases of suspected human infection with the influenza A(H7N9) virus and in the surveillance of both avian and human influenza viruses. A diagnostic system using this assay was prepared at 74 prefectural and municipal public health institutes and 16 quarantine stations in Japan early into the human H7N9 infection outbreaks, enabling potential diagnoses of H7N9 infection across Japan.

Development and evaluation of a new real‐time RT‐PCR assay for detecting the latest H9N2 influenza viruses capable of causing human infection

“…In our previous study, we developed rRT‐PCR assays to detect types of influenza A and B viruses, determine subtypes of H1pdm09, former H1 (Russian flu), H3, H5 and H7 influenza A viruses, and discriminate between the Victoria and Yamagata lineages of influenza B viruses. These assays can be performed under the same conditions as the H9 assay described in the present report (http://www.who.int/influenza/gisrs_laboratory/molecular_diagnosis/en/) . Hence, by combining these methods, influenza viruses can be easily and simultaneously identified with respect to type and subtype or lineage with high sensitivity for diagnostic and monitoring purposes.…”

“…Viral RNA was extracted from 140 μL cultures of each virus propagated in embryonated chicken eggs to 60 μL of AVE (elution buffer supplied with the kit) using a QIAamp ® viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. In this study, the copy number of the M gene of the 18 H9 viruses was determined quantitatively as previously described using an influenza A (type A) rRT‐PCR assay targeted to the universal M gene of all influenza A viruses . The threshold cycle (Ct) values of viral RNA extracted from 19 viral respiratory pathogens were determined using the multiplex real‐time PCR assay described previously, with minor modifications .…”

Detection of influenza A virus from live-bird market poultry swab samples in China by a pan-IAV, one-step reverse-transcription FRET-PCR

“…The sensitivities and specificities of PCR-based methods are critically determined by the choice of the sequences for primers and probes 23 24 . The sequences for the primers and/or probes which were described earlier for IAV detection may be appropriate for some IAV subtypes, or strains only circulating in humans or poultry, but have many mismatches to IAV strains found in other animals 11 12 13 14 15 16 17 18 19 20 . In this study, we used the extensive sequence information available for IAV in GenBank to design a pan-IAV primer and probe set.…”

“…Traditionally, the gold standard for IAV detection involves virus replication in eggs or tissue culture followed by HA inhibition and NA inhibition assays 9 10 11 . While the virus propagation in egg and inhibition assays are laborious and not very sensitive, many powerful reverse transcription-PCR and real-time RT-PCR assays have been successfully established for IAV detection 10 11 12 13 14 15 16 17 18 19 20 . However, to the best of our knowledge, there is no real-time one-step RT-PCR test which can conveniently detect most IAV subtypes in clinical samples.…”

Development and Evaluation of New Real-Time RT-PCR Assays for Identifying the Influenza A Virus Cluster IV H3N2 Variant

“…Although human-like H3 has become the predominant H3N2 among swine in the USA, Cluster IV H3N2v is still maintained; that is, the threat to public health associated with Cluster IV H3N2v remains (10). Our previously developed rRT-PCR assays for discriminating influenza virus types, subtypes, and lineages can be performed under the same conditions as the assays developed in this study (11,(13)(14)(15). Hence, by combining the previous and newly developed assays, Cluster IV H3N2v can be directly identified with high sensitivity and specificity for diagnosis and monitoring without sequencing the H3 HA gene.…”