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