Development of a loop-mediated isothermal amplification for rapid diagnosis of Aphelenchoides ritzemabosi

To establish the amplification and detection system for the chrysanthemum foliar nematode (CFN), Aphelenchoides ritzemabosi, the loop-mediated isothermal amplification (LAMP), which includes two external primers (F3/B3), two inner primers (FIP/BIP) and one loop primer (LF), was designed based on the CFN 18S ribosomal RNA gene. The DNA samples were amplified by LAMP primers using isothermal amplification (65 °C). The amplified products were detected by electrophoresis and visual inspection of fluorescent dyes. Detection of CFN in a sample was confirmed when the electrophoresis results show a DNA ladder or the mixtures in the tubes showed green fluorescence. The results showed that the LAMP method could not only detect and identify a single female, male, juvenile and egg of CFN but also directly detect CFN in mixed nematode species samples and plant tissue samples. The sensitivity of the LAMP assay was 100-fold dilution of single nematode DNA.