Abstract:
Objective To establish a detection assay based on digital droplet polymerase chain reaction (ddPCR) for the quantitative and accurate detection of Brucella nucleic acid in the blood of brucellosis patients.
Methods By using Brucella Bcsp31 gene as the target gene, the primers and probe were designed and the recombinant positive plasmid pUC57-Bcsp31 was constructed. The sensitivity, repeatability and specificity of ddPCR assay for detection of Brucella were evaluated. In addition, 120 blood samples were collected from suspected brucellosis patients, and ddPCR was used to quantitatively detect Brucella DNA load in the samples.
Results The ddPCR assay established in this study had high sensitivity and the minimum detection limit was 100 copies /μL, and showed good specificity and accuracy for the detection of Brucella. Of the 120 blood samples tested, 103 were positive by ddPCR, and 52 were positive by quantitative real-time PCR (qPCR). In 105 antibody positive samples, 93 and 51 were detected to be positive by ddPCR and qPCR, and the positive rates were 88.57% and 49.52%, respectively. In the 15 antibody negative samples, 12 and 1 samples were detected positive by the two assays, respectively.
Conclusion This study established a detection assay with high sensitivity and high specificity for Brucella detection based on ddPCR, which can be used for the quantitative and accurate detection of Brucella nucleic acid in blood samples of brucellosis patients. It is of great significance for early screening of brucellosis in suspected patients with negative antibody test results.
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