Application of real-time PCR in detection of Brucella / 中华地方病学杂志

ABSTRACT

Objective To establish a real-time quantitative PCR (RT-PCR) preliminary screening method for rapid detection of Brucella.Methods Based on the nucleotide sequence encoding Brucella's outer membrane proteins omp10 and omp31,four primers and probes of omp10,omp10-1,omp31 and omp31-1 were designed.The primers and probes were used to detect 19 strains of 6 categories of Brucella DNA of known organisms,10 strains of Brucella DNA that were isolated from Yuxi of Yunnan.And 224 negative Brucella DNA,including 35 strains of Bartonella DNA,103 strains of the Lord Komori enterocolitis DNA (including 4 parts of O ∶ 3 and 9 parts of O ∶ 9) and 86 samples of hybrids bacteria DNA.Then the specificity of primers and probes were evaluated based on the test results.Results The DNA of 19 standard Brucella strains could be amplified by omp10 and omp10-1,and the peak time and amplification curve of omp10 is better than omp10-1,and the DNA of negative control strains could not be amplified by omp10.The DNA of 16 standard Brucella strains could be amplified by omp31-1.The DNA of standard Brucella strains could not be amplified by omp31.The average Ct values of 10 strains of Brucella DNA which were isolated from Yuxi of Yunnan that were detected by omp10,omp10-1 and omp31-1 respectively were 19.87,19.14 and 17.52.Conclusion Omp10 has strong specificity and only specific for Brucella,so it can be used for rapid detection of Brucella.