Abstract
A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus.
Highlights
Avian influenza (AI) is a highly contagious avian influenza disease caused by the orthomyxovirus class A influenza virus, with 16 HA subtypes and 10 NA subtypes [1]
Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR
DRT-PCR was established by comparing the sensitivity, specificity and reproducibility of dRT-PCR and real-time quantitative RT-PCR, and it was applied to the detection of actual samples, so as to achieve rapid detection of H9 subtype low pathogenic avian influenza
Summary
Avian influenza (AI) is a highly contagious avian influenza disease caused by the orthomyxovirus class A influenza virus, with 16 HA subtypes and 10 NA subtypes [1]. According to the avian influenza virus (Avian influenza virus, AIV), pathogenicity can be divided into highly pathogenic avian influenza and low pathogenic avian influenza virus [2]. As a novel nucleic acid detection method, digital PCR (dPCR) technology independently performs PCR reactions by dividing the reaction system into a large number of reaction units and calculating the number of nucleic acids according to the Poisson distribution and the positive ratio [4]. Unlike traditional quantitative PCR techniques, dPCR is independent of the circulatory threshold (CT) of the amplification curve and is not affected by amplification efficiency, with good accuracy and reproducibility, and can achieve absolute quantitative analysis [5]. DRT-PCR was established by comparing the sensitivity, specificity and reproducibility of dRT-PCR and real-time quantitative RT-PCR (qRT-PCR), and it was applied to the detection of actual samples, so as to achieve rapid detection of H9 subtype low pathogenic avian influenza
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