Subpopulation Primers Essential for Exhaustive Detection of Diverse Hemagglutinin Genes of H5 Subtype Avian Influenza Viruses by Loop-Mediated Isothermal Amplification Method

Journal of Clinical Microbiology
Y FuruyamaKenji Tsukamoto

Abstract

Loop-mediated isothermal amplification (LAMP) is a potential screening test for avian influenza (AI), but its narrow detection spectrum limits its applications. To improve this narrow detection spectrum, 3 types of primers were compared for detection of diverse H5 subtype hemagglutinin (HA) genes. Four and 6 genes, of 10 genetically different H5 HA genes tested, were detected with S primers specific for A/duck/Tsukuba/9/2005 (H5N2) and with M primers (which contained mixed bases), respectively. In contrast, all 10 HA genes became positive with population primers (P primers) (a mixture of primers designed for each subpopulation of 2,202 HA genes). Our study indicated that the P primers for the forward inner primer (FIP) and backward inner primer (BIP) sites were essential for exhaustive detection, whereas those for the F3, forward loop (FL), backward loop (BL), and B3 sites were exchangeable with M primers. A base mismatch experiment demonstrated that HA genes with ≤2 base mismatches per primer site and ≤10 base mismatches per HA gene were amplifiable. Reverse transcription-LAMP was broadly reactive, specific for H5 subtype HA genes, and applicable to field samples, with the sensitivity of real-time PCR. The in silico analysis s...Continue Reading

References

Jun 8, 2007·Emerging Infectious Diseases·Shanthi JayawardenaLeo L M Poon
May 1, 2009·Journal of Clinical Microbiology·Kenji TsukamotoMasaji Mase
May 31, 2013·Clinical Microbiology and Infection : the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases·K NieX J Ma
Jul 12, 2013·Nature·Tokiko WatanabeYoshihiro Kawaoka
Nov 1, 2015·Transboundary and Emerging Diseases·F J ConrathsT Homeier-Bachmann

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