OPTIMIZATION OF THE POLYMERASE CHAIN REACTION (PCR) METHOD FOR THE DETECTION OF Oryctes rhinoceros VIRUS

Optimization of the polymerase chain reaction (PCR) method for the rapid detection of Oryctes rhinoceros virus (OrV) was studied. The virus DNA was extracted from the gut tissues by a robust method. Using a pair of specific primers, Primer 15a and 15b, infection was confirmed when the PCR product produced a single 945 bp DNA band. The optimized concentrations of the PCR components were at 2.0 mM MgCl2, 1.0 mM 10X PCR buffer, 0.2 mM Primer 15a and 15b, 0.5 U Taq-DNA polymerase and 0.4 mg bovine serum albumin (BSA). All tested virus DNA concentrations at 0.085, 0.170 and 0.255 mu g mu l(-1) were suitable for virus detection. Addition of BSA (20 mg ml(-1)) at 0.4 mg in the reaction increased the PCR sensitivity. The method is capable of detecting OrV infection from DNA diluted one million times or equivalent to a virus DNA concentration as low as 2.23 pg mu l(-1). The PCR detected 83.2% adult beetles from pheromone traps as being infected by OrV, 13.6% higher (P<0.05) than the results based on observations on the gut morphological appearance (69.6%). A typical OrV infection symptom is a swollen gut filled with milky fluid. Of the 839 guts with this symptom, 97.6% were diagnosed to be infected which was not significantly different (P>0.05) compared to the method based on gut morphological appearance. The PCR was also capable in detecting virus at an early infection stage and in dead adults with decayed tissues. Of the 307 adults that appeared to be healthy, 36.1% of them were found to be infected. As much as 61.6% of dead adults with decayed tissues (N = 428) were diagnosed to be infected by the OrV. The method can be used in further research studies relating to OrV for the management of the rhinoceros beetle.