Evaluation of detection of koi herpesvirus disease by conventional polymerase chain reaction (PCR)

Abstract

The method of detection of koi herpesvirus disease continues to be developed to obtain a high level of sensitivity and specificity. One common test method uses conventional PCR. This method resulting not only relatively inexpensive cost analysis but also rapid analysis. This experiment is using four types of commercial kits, the primers design of TK and Sph as well as some annealing temperatures. The sample used was the result of DNA extraction from koi gills, positive control of the collection of KHV 0301 FRA, positive control KHV with dilution concentration of 10000; 1000; 100; 10 and 1, to regulate the presence of contamination on amplification process, a negative control must be included, ddH2O. The annealing temperatures used are 52 0C; 55 0C; 56 0C; 60 0C and 63 0C. This experiment showed different results of KHV detection. The sensitivity and specificity of the results are different for each kit, primers pairs and annealing temperature. Using a one-degree difference in annealing temperature will affect the detection of KHV although using the same kind of kits and primer pairs. The user is obliged to do optimization method regarding the kits usage, primers pairs and various temperatures to obtain a diagnostic method that has high level sensitivity and specificity detection. The sensitivity detection of KHV virus in this experiment could detect up to 10 copies. The user or analyst have to know and understand about all the information in the manual kits.