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| Rapid and Sensitive Detection of Meloidogyne camelliae by LAMP-LFD |
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Abstract Aiming at improving the detection efficiency, a novel loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for rapid detection of Meloidogyne camelliae was developed, mostly based on the nucleotide amplification by a LAMP and visual detection by a LFD. Three pairs of primers were designed targeting the conserved region of ribosomal DNA-internal transcribed spacer (rDNA-ITS) and used in the biotinylated Real-time fluorescence LAMP. The optimized LAMP was of 63 ℃ 15 min. LFD, Real-time fluorescence curve and agarose gel electrophoresis were separately used for LAMP products detection, and the practicability of LFD was comparatively evaluated. The results indicated that LAMP products were hybridized by a fluorescein isothiocyanate (FITC)-labeled probe ITS-HP and visually detected in LFD in 5 min. It just needed 25 min from the beginning of LAMP reaction to the judgment of results, which was approximately 2 h time-savings compared with the conventional PCR method. The results demonstrated that LAMP-LFD could specifically detect M. camelliae with a detection limit of 4 pg/μL M. camelliae genomic DNA, which was lower than the conventional PCR of 100 times, and the detection sensitivity was 1/1 000 for a single juvenile J2 genomic DNA. Therefore, this rapid, sensitive and specific LAMP-LFD was an expected option in the inspection and quarantine for M. camelliae.
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Received: 08 October 2015
Published: 01 April 2016
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