Optimization and evaluation of loop-mediated isothermal amplification (LAMP) system for detection of Pseudocercospora angolensis in citrus cinensis

Abstract

Pseudocercospora angolensis causes Pseudocercospora leaf and fruit spot disease in citrus which can result in up to 100% yield loss. Early diagnosis of this disease is vital for effective control. This study aimed at optimizing and evaluating a loop-mediated amplification (LAMP) system for detecting P. angolensis in sweet oranges using microscopy as a gold standard. The analytical specificity and sensitivity of PCR and LAMP were assessed using twelve (12) non-target species and serial dilutions of P. angolensis DNA respectively. The diagnostic accuracies of the two assays were evaluated using 150 diseased and 50 non-diseased sweet orange leaf samples. The analytical sensitivity and detection time of LAMP were 10-4 ng/µl and 40 minutes respectively. The analytical sensitivity of PCR was 10 ng/µl and it was specific to P. angolensis whereas three relatives of P. angolensis were detectable by LAMP. The diagnostic sensitivities of LAMP (93%) and microscopy (100%) were not significantly different (X2 = 8.38, P = 0.0038) unlike the diagnostic specificities (90%) and (100%), respectively (X2 = 3.37, P = 0.066). Microscopy was significantly more sensitive than PCR (32.6%) (X2 = 149.26, P < 2.2e-16) and equally specific as PCR (P=NA). The positive predictive values of PCR and LAMP were 100% and 96.5% respectively whereas the negative predictive values were 33.1% and 81.8% respectively. The high positive and negative predictive values of LAMP make it a more suitable assay for screening sweet orange samples for P. angolensis than PCR.