Subpopulation Primer Strategy Achieves Exhaustive H5 Avian Influenza Detection by LAMP
A novel subpopulation primer design for loop-mediated isothermal amplification enabled detection of all tested genetically diverse H5 hemagglutinin genes, overcoming LAMP's traditionally narrow detection spectrum. In silico analysis predicted coverage of 99.9% of H5 sequences in GenBank. The approach provides real-time PCR-equivalent sensitivity in an isothermal format suitable for general diagnostic laboratories conducting avian influenza surveillance.
The original study
Subpopulation Primers Essential for Exhaustive Detection of Diverse Hemagglutinin Genes of H5 Subtype Avian Influenza Viruses by Loop-Mediated Isothermal Amplification Method.
- Authors
- Furuyama Y, Takahashi Y, Noguchi K, Murakami H, Sakaguchi M, Hisamatsu S, et al.
- Journal
- Journal of clinical microbiology
- Type
- Journal Article, Research Support, Non-U.S. Gov't, Validation Study
- PMID
- 30021821
Original 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 suggested that most H5 HA genes (2,586 positive genes/2,588 genes tested) registered in the GenBank database might be amplifiable. These results indicate that the use of subpopulation primers in LAMP allows exhaustive detection of diverse HA genes and H5 LAMP can be used as a reliable AI screening test in general diagnostic laboratories.