Development of a loop-mediated isothermal amplification for rapid diagnosis of Aphelenchoides ritzemabosi
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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.
KeywordsAphelenchoides ritzemabosi Loop-mediated isothermal amplification 18S ribosomal RNA Single nematode Mixed samples
This work was supported by National Natural Science Foundation of China (No. 31871939), Key science and technology project of Hainan Province (No. 080151) and Special research and development of Applied Technology in Hainan (No. ZDXM2014020).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This study does not contain any studies with human participants or animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
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