Hapten-labeled fusion-polymerase chain reaction of multiple marker genes for the application of immunochromatographic test.

A variety of methods have been reported using polymerase chain reaction (PCR)-based nucleic acid testing (NAT) because of its potential to be used in highly sensitive inspection systems. Among these NATs, fusion-PCR (also called as overlap-extension-PCR) has been focused on this study and adopted to generate the fused amplicon composed of plural marker gene fragments for detection. Generally, conventional agarose gel electrophoresis followed by gel staining is employed to check the PCR results. However, these are time-consuming processes that use specific equipment. To overcome these disadvantages, the immunochromatographic test (ICT) for the detection of PCR amplicons with hapten-labels that were generated by PCR using hapten-labeled primers was also adopted in this study. Based on these concepts, we constructed the systems of hapten-labeled fusion-PCR (HL-FuPCR) followed by ICT (HL-FuPCR-ICT) for the two and three marker genes derived from pathogenic microbe. As a result, we successfully developed a two marker genes system for the pathogenic influenza A virus and a three marker genes system for the penicillin-resistant Streptococcus pneumoniae. These detection systems of HL-FuPCR-ICT are characterized by simple handling and rapid detection within few minutes, and also showed the results as clear lines. Thus, the HL-FuPCR-ICT system introduced in this study has potential for use as a user-friendly inspection tool with the advantages especially in the detection of specific strains or groups expressing the characteristic phenotype(s) such as antibiotic resistance and/or high pathogenicity even in the same species.