PCR optimization and evaluation for molecular detection of Renibacterium salmoninarum in rainbow trout (Oncorhynchus mykiss)

Document Type: Primary Research paper

Authors

Abstract

The goal of this study was to optimize the molecular polymerase chain reaction technique (PCR) to detect R. salmoninarum, as well to evaluate and determine the sensitive limits, specificity, and predictive values. The methodology comprised two phases: first, was the implementation and optimization of nested PCR technique to detect R. salmoninarum, and second, was the technique evaluation through sensitive and specificity tests. In second phase, it was needed to make cycling and temperature modifications of primers alignment to optimize and suppressed to unspecific bands. Tested primers were P3, M21, P4, and M38 against two species phylogenetically associated: A. salmonicida and M. luteus. Decimals dilutions were applied to sensitive test. The calculated specificity was 100%, with positive predictive value of 100%, and negative value of 100%. With respect sensibility, based in 12.1 ng µL-1 concentration, was detected (0.121 ng µL-1) in first step, and 1.21 × 10-4 ng µL-1 at second step. While at a 26.1 ng µL-1 concentration, was detected 2.61 × 10-3 ng µL-1 in first step, and 2.61 × 10-10 ng µL-1. We can conclude that nested PCR technique was selective and effective for estimated detection of 6.5 cells of R. salmoninarum.

Keywords