Abstract
Avian influenza (AIV) outbreaks can induce fatal human pulmonary infections in addition to economic losses to the poultry industry. In this study, we aimed to develop a rapid and sensitive point-of-care AIV test using loop-mediated isothermal amplification (LAMP) technology. We designed three sets of reverse transcription LAMP (RT-LAMP) primers targeting the matrix (M) and hemagglutinin (HA) genes of the H5 and H9 subtypes. RT-LAMP targeting the universal M gene was designed to screen for the presence of AIV and RT-LAMP assays targeting H5-HA and H9-HA were designed to discriminate between the H5 and H9 subtypes. All three RT-LAMP assays showed specific amplification results without nonspecific reactions. In terms of sensitivity, the detection limits of our RT-LAMP assays were 100 to 1,000 RNA copies per reaction, which were 10 times more sensitive than the detection limits of the reference reverse‒transcription polymerase chain reaction (RT-PCR) (1,000 to 10,000 RNA copies per reaction). The reaction time of our RT-LAMP assays was less than 30 min, which was approximately four times quicker than that of conventional RT-PCR. Altogether, these assays successfully detected the existence of AIV and discriminated between the H5 or H9 subtypes with higher sensitivity and less time than the conventional RT-PCR assay.
Highlights
Influenza A viruses, which are classified into 18 hemagglutinin (HA) and 11 neuraminidase subtypes based on their antigenic properties, have been found to infect avian species, and multiple mammalian hosts including humans
We developed RT-loop-mediated isothermal amplification (LAMP) assays that can detect a universal target for avian influenza viruses (AIVs) and can discriminate between the H5 and H9 subtypes of AIV
Primer design and optimization of reverse transcription LAMP (RT-LAMP) assays For RT-LAMP, primers were designed for the M, H5-HA, and H9HA genes
Summary
Influenza A viruses, which are classified into 18 hemagglutinin (HA) and 11 neuraminidase subtypes based on their antigenic properties, have been found to infect avian species, and multiple mammalian hosts including humans. Avian influenza viruses (AIVs) belong to the category of influenza A viruses, which induce human infections and cause economic losses annually [1,2,3]. The outbreaks spread widely throughout the globe [4], and caused several human infections, including fatal cases [5]. In addition to HPAIVs, low-pathogenic avian influenza viruses (LPAIVs), which usually produce mild or no symptoms in birds, can induce pathogenic avian influenza by antigenic drift or shift, and can induce human infection [2]. Identification of HPAIVs and LPAIVs would be important to control avian influenza and potential human pandemic infections
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