In this study, we developed new sets of primers to detect
Brucella spp. and M.
avium subsp. paratuberculosis (MAP)
through isothermal amplification. We selected a previously well-characterized
target gene, bscp31, specific for Brucella
spp. and IS900 for MAP. The limits of detection using the
loop-mediated isothermal amplification (LAMP) protocols described herein were
similar to those of conventional PCR targeting the same sequences.
Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye
detection with identical sensitivity as agarose gel electrophoresis. We included
the LAMP-based protocol in a rapid identification scheme of the respective
pathogens, and all tested isolates were correctly identified within 2 to 3 h. In
addition, both protocols were suitable for specifically identifying the
respective pathogens; in the case of Brucella, it also allowed
the identification of all the biovars tested. We conclude that LAMP is a
suitable rapid molecular typing tool that could help to shorten the time
required to identify insidious bacteria in low-complexity laboratories, mainly
in developing countries.
Aim:This study aimed to compare the sensitivity (S), specificity (Sp), and positive likelihood ratios (LR+) of four polymerase chain reaction (PCR) assays for the detection of Brucella spp. in dog’s clinical samples.Materials and Methods:A total of 595 samples of whole blood, urine, and genital fluids were evaluated between October 2014 and November 2016. To compare PCR assays, the gold standard was defined using a combination of different serological and microbiological test. Bacterial isolation from urine and blood cultures was carried out. Serological methods such as rapid slide agglutination test, indirect enzyme-linked immunosorbent assay, agar gel immunodiffusion test, and buffered plate antigen test were performed. Four genes were evaluated: (i) The gene coding for the BCSP31 protein, (ii) the ribosomal gene coding for the 16S-23S intergenic spacer region, (iii) the gene coding for porins omp2a/omp2b, and (iv) the gene coding for the insertion sequence IS711.Results:The results obtained were as follows: (1) For the primers that amplify the gene coding for the BCSP31 protein: S: 45.64% (confidence interval [CI] 39.81-51.46), Sp: 95.62% (CI 93.13-98.12), and LR+: 10.43 (CI 6.04-18); (2) for the primers that amplify the ribosomal gene of the 16S-23S rDNA intergenic spacer region: S: 69.80% (CI 64.42-75.18), Sp: 95.62 % (CI 93.13-98.12), and LR+: 11.52 (CI 7.31-18.13); (3) for the primers that amplify the omp2a and omp2b genes: S: 39.26% (CI 33.55-44.97), Sp: 97.31% (CI 95.30-99.32), and LR+ 14.58 (CI 7.25-29.29); and (4) for the primers that amplify the insertion sequence IS711: S: 22.82% (CI 17.89 - 27.75), Sp: 99.66% (CI 98.84-100), and LR+ 67.77 (CI 9.47-484.89).Conclusion:We concluded that the gene coding for the 16S-23S rDNA intergenic spacer region was the one that best detected Brucella spp. in canine clinical samples.
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