Amplifikasi gen penyandi hemolysin dari bakteri vibrio harveyi dengan teknik PCR (Polymerase Chain Reaction)

Abstract

Vibriosis disease which is caused by lumiunescent Vibrio harveyi until today become a trouble in the cultivation of vannamei shrimp. When the epidemic occurs and the density of bacterial population reachs in thousands times, it causes the mass mortality of the shrimp larvae. Therefore the diagnose method that can accurately and quickly detect this bacterial pathogen is absolutely required. One of method that commonly used is PCR (polymerase chain reaction)-based method. The objective of this research was to evaluate the primer pair application targeted the hemolysin gene, so that primer pair which has been designed can successfully amplify and therefore can detect the presence of hemolysin gene in V. harveyi. This primer pair was designed from the local vannamei strain isolated from Indonesia. Eight vibrio isolates obtained from shrimp, were used in this study. After the cell morphologically tested with Gram staining, then the following methods were applied as follows ; optical density count, hemolysin test, DNA extraction and PCR method optimalization. The result of blood hemolysis test showed that all of V. harveyi isolates produced clear zones surround their colonies on blood agar medium. The PCR result using Myhemo primer pair of F1 5’-GATGGTCAGTGCCTCTCA-3’(forward) and R1 5’- CCCAGTTGTATAGCGGTA-3’(reverse) successfully amplified the hemolysin gene of isolates V-U5 and V-U24 in size of approximately of 518 bp. The PCR programme used in this study was at the following conditions ; DNA denaturation at 94oC for 1 min, primer annealing at 50oC for 1 min and DNA extension at 72 oC for 1min as many as 35 cycles.