Detection of human influenza A viruses by loop-mediated isothermal amplification

Abstract

Here we describe the use of the loop-mediated isothermal amplification (LAMP) method to detect human influenza viruses (H1 to H3). Our results were correlated 100% with results deduced from routine clinical diagnostic tests. In addition, we also developed a LAMP assay specific for human beta-actin cDNA as a quality control test.

FIG. 1.

LAMP assay for human influenza A virus. (A) Primers and representative M gene sequence used in the assay. The first and last nucleotides of the sequence are the 369th and 609th nucleotides, respectively, of the referenced sequence used in this study (GenBank accession number L25818). Locations of primer binding sequences are indicated by arrows. The FspI restriction sites are bolded. (B) Detection limits of LAMP (upper panel) and PCR (lower panel) assays. Serial diluted cDNA derived from influenza A/WSN/33 was tested by these assays. The concentrations of cDNA used in these reactions are indicated (lanes 1 to 14). (C) Detection of different human influenza A viruses. Lane 1, DNA markers; lane 2, no template control; lane 3, H1 subtype lane 4, H2 subtype; lane 5, H3 subtype.

FIG. 2.

LAMP assay for β-actin. (A) Primers and β-actin sequence used in the assay. The first and last nucleotides of the sequence are the 300th and 599th nucleotides, respectively, of the referenced sequence in this study (GenBank accession number NM_001101). Corresponding recognition sequences of the primers are indicated by arrows. The BseYI and MboI restriction sites are boldfaced and italicized, respectively. Apart from the primer sequences, the reaction condition was identical to the one for the detection of influenza viruses. (B) Detection of β-actin sequences in NPA samples. Lane 1, DNA markers; lane 2, influenza A virus; lane 3, human metapneumovirus; lane 4, respiratory syncytial virus; lane 5, adenovirus; lane 6, SARS coronavirus; lane 7, rhinovirus; lane 8, influenza B virus.