Validation Of Colletotrichum asianum Detection: A Comparative Study Between PUP DETECT LAMP Assay Kit And Conventional PCR

Alethea Vlen Lopez CS-4 Fungal genetics, genomics and bioinformatics

Validation of Colletotrichum asianum Detection: A Comparative Study between PUP DETECT LAMP Assay Kit and Conventional PCR

Alethea Vlen M. Lopez1*, Rommel P. Aban1, Abigail O. Indong1, Lourdes V. Alvarez1, and Chester C. Deocaris2

1 Department of Biology, College of Science, Polytechnic University of the Philippines, Sta. Mesa, Manila, 1008 Philippines

2 Department of Physical Sciences, College of Science, Polytechnic University of the Philippines, Sta. Mesa, Manila, 1008 Philippines

*Email: avmlopez14@gmail.com

The PUP DETECT LAMP Assay Kit is a species-specific assay to detect Colletotrichum asianum in Philippine Carabao Mango (Mangifera indica L. var. Carabao, CM). This study validates the detection of C. asianum in CMs and compares the diagnostic accuracy of the LAMP assay to conventional PCR. PCR optimization using isolated C. asianum culture and the F3 and B3 primers from the PUP DETECT LAMP Assay Kit determined the optimal annealing temperature at 56°C and MgCl2 concentration of 2.50 mM. CMs with 50% anthracnose prevalence were collected and subjected to both amplification tools. LAMP detected seven positive C. asianum amplifications in symptomatic CMs (SCM) and two positive amplifications in PCR. All asymptomatic CMs (ACM) tested negative in both assays. LAMP exhibited greater diagnostic sensitivity (Se) of 88% than PCR’s 25%, while both assays demonstrated 100% diagnostic specificity (Sp). LAMP also revealed a higher diagnostic NPV (97%) than PCR (84%), with both assays having the same diagnostic PPV (100%). The analytical Se and Sp of both assays were also determined. LAMP declared a higher analytical Se detecting fungal DNA at a level as low as 1.165×10-9 pg, compared to PCR’s limit of detection at 2.329 pg. Both assays have the same analytical Sp, solely detecting C. asianum. The PUP DETECT LAMP Assay Kit is a more sensitive and simpler method for detecting C. asianum in CMs compared to conventional PCR. It enables rapid and sensitive detection of C. asianum, facilitating early identification of anthracnose disease in CMs and reducing post-harvest losses.