Systematic evaluation of TaqMan real-time polymerase chain reaction assays targeting the and loci of in recombinant plasmids and naturally infected dogs.

BACKGROUND AND AIM

Because of the diversity of local genotypes of , genes targeted by TaqMan real-time polymerase chain reaction (RT-PCR) assays should be systematically evaluated. This study evaluated the amplification efficiency, linearity, precision, and sensitivity of two TaqMan RT-PCR assays targeting the and loci of in recombinant plasmids and naturally infected dogs.

MATERIALS AND METHODS

Thirty blood samples were collected from dogs showing clinical signs of canine monocytic ehrlichiosis at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand. The and genes were amplified by conventional PCRs (cPCRs) on the blood samples and were then sequenced. Meanwhile, RT-PCR was used to detect and genes in 10-fold dilutions of the recombinant plasmids.

RESULTS

Both and were amplified with a high degree of linearity ( ≥0.975 and 0.993, respectively) in all dilutions, although the mean percentage of relative standard deviation of A was lower, but the difference was non-significant. The detection limits of RT-PCR and cPCR were 10 and 10, respectively, for both loci. RT-PCR targeting (22/30; 73.3%) and (15/30; 50%) yielded a number of positive results that did not differ significantly (p=0.06). The RT-PCR positive results of the gene (22/30) differed significantly from that of cPCR (11/30) (p=0.004). In contrast, the RT-PCR positive results of the gene (15/30) did not differ significantly from that of cPCR (12/30) (p=0.43). The mean Ct value (30.2) based on RT-PCR of 22 positive cases was higher than that of RT-PCR (Ct=27.4) on 15 positive cases. The Ct values from RT-PCR were >30 in all seven discordant samples that were not detected by the RT-PCR.

CONCLUSION

RT-PCR targeting the gene was more sensitive for detecting in naturally infected dogs. This study suggested that TaqMan RT-PCR of the gene should be selected for research in this region.