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| Detection of H9 avian influenza virus by Loop-mediated Isothermal Amplification and Application of Fluorescent Reagent |
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Abstract Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was used to develop a specific, sensitive and convenient method for detection of H9 subtype of Avian influenza virus(H9-AIV). Six primers directing to eight recognition sites on the hemagglutinin(HA) gene of H9-AIV were used, and at least as 103 copies of the target gene fragment could be detected after reaction for 60 minutes at 63℃, with a sensitivity of 10-fold higher than that of RT-PCR. High species-specificity of the RT-LAMP method was confirmed by the assay of H1~H15 hemagglutinin(HA) subtypes Avian influenza viruses, Newcastle disease virus(NDV) and Infectious bronchitis virus(IBV). A mixture of calcein and Mn2+ was used as fluorescent reagent to make the result of RT-LAMP visible, and similar results could be obtained as the turbidity test. Furthermore, 109 clinical samples were assayed by RT-LAMP and RT-PCR, of which 61 and 46 samples were confirmed as positive by the RT-LAMP and RT-PCR, respectively. So we could draw a conclusion that the sensitivity of RT-LAMP was preferable to the RT-PCR assay.
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Received: 07 May 2010
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