PCR Validation of PUP Detect – LAMP Diagnostic Kit for Stem End Rot Disease in Philippine Mango var. “Carabao”
Regine S. Ibrahim1*, Ralph Niño Mecaydor1, Myra Angela A. Pangilinan1, Chester C. Deocaris2 and Lourdes V. Alvarez1
1Department of Biology, College of Science, Polytechnic University of the Philippines – Manila, Philippines
2Department of Physical Science, College of Science, Polytechnic University of the Philippines – Manila, Philippines
Mangifera indica Linn. cv ‘Carabao’ is one of the significant fruit crops in the Philippines, both in domestic and export markets. Lasiodiplodia theobromae, the major causative fungus of mango stem-end rot (SER) disease, causes a significant reduction in mango yield production. A recent study by Alvarez et al. (2020) established a loop-mediated isothermal amplification (LAMP) kit designed to detect SER disease in Philippine ‘Carabao’ mango. The study aimed to validate the LAMP assay kit developed by Alvarez et al. to detect the RPB2 gene of L. theobromae through comparative analysis with conventional PCR. Analytical and diagnostic testing was performed to confirm the limit of detection (LOD) and accuracy of the LAMP assay. LAMP reaction was performed under 63.8 °C for 38 minutes while PCR reaction was performed at 56.5⁰C optimized annealing temperature and 2 mM MgCl2 concentration. The analytical specificity was confirmed by using L. theobromae positive control, negative controls (5 non-L. theobromae DNA), and blank control. Analytical sensitivity test showed that the LAMP has LOD of 9.75×10-7 fg lower than the 195 fg LOD of the PCR assay. Diagnostic testing showed that LAMP assay was highly sensitive in detecting the L. theobromae compared to the conventional assay, whereas the specificity as well as the PPV, are lower in contrast to the values of PCR due to the false positives. The study validated that the LAMP assay based on gene RPB2 appeared to be rapid and simple and could also be used to identify L. theobromae from Philippine ‘Carabao’ mango.