Molecular Diagnosis of Bordetella spp. by Means the Semi-Nested Polymerase Chain Reaction
Traditionally, the diagnosis of Bordetellabron chiseptica (B. bronchiseptica) is based mainly on clinical signs and epidemiological history. Although the bacteriological culture has diagnostic specificity, it is a technique where the microorganisms present slow development and where the phenotypic identification of the bacteria is complex. On the other hand, its sensitivity could be lower when compared with molecular diagnostic, since only a fraction of all bacteria can grow in an artificial environment. Therefore, implementing diagnostic techniques of high sensitivity and specificity can contribute to the detection and management associated with respiratory symptoms produced by this agent. The objective of this work is to implement a diagnostic protocol for Bordetella spp. by the semi-nested Polymerase Chain Reaction (sn-PCR)by means the detection of the gene which encodes the structural protein flagellin. For this, three in silico primers were designed using the free access Oligo Perfect ™ program, which was later synthesized bythe BIOSCAN company. Thus, using three strains of B. bronchisepticaas samples, fragments of the expected sizes were obtained (362 bp and 170 bp) and, after the sequencing of the larger amplicon, a percentage of 95% nucleotide identity was determined by the Clustal Ω program with respect to the official data registered in GenBank®. This value was corroborated when entering the same sequence to the BLAST online program, which also delivered 95% nucleotide identity percentage concerning to the flagellin protein gene of Bordetella spp.
