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Evaluation of loop‐mediated isothermal amplification (LAMP) assays based on 5S rDNA‐IGS2 regions for detecting Meloidogyne enterolobii
Author(s) -
Niu J. H.,
Jian H.,
Guo Q. X.,
Chen C. L.,
Wang X. Y.,
Liu Q.,
Guo Y. D.
Publication year - 2012
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1111/j.1365-3059.2011.02562.x
Subject(s) - loop mediated isothermal amplification , biology , dipstick , amplicon , sybr green i , microbiology and biotechnology , detection limit , genomic dna , dna , polymerase chain reaction , chromatography , genetics , gene , chemistry , biochemistry , urine
A loop‐mediated isothermal amplification (LAMP) assay for detection of Meloidogyne enterolobii (Me‐LAMP) was developed based on the sequences of the 5S ribosomal DNA (5S rDNA) and intergenic spacer 2 (IGS2) segment. The LAMP amplification was achieved at 65°C isothermal conditions within 1–1·5 h. Its amplicons were confirmed using gel electrophoresis, Sac I enzyme analysis, lateral flow dipstick (LFD) assay, and visual inspection through SYBR Green I and calcein staining. The results demonstrated that the Me‐LAMP was able to specifically detect M. enterolobii populations from different geographical origins, with a detection limit of about 10 fg M. enterolobii genomic DNA, which was 10–100 times more sensitive than conventional PCR. In addition, the applicability of LAMP to field detection was confirmed following its successful performance in detecting the pest on root and soil samples. The Me‐LAMP assay possessed the characteristics of simplicity, sensitivity and specificity, and is a promising and practical molecular tool for M. enterolobii diagnosis in pest quarantine and field surveys.