Establishment of a nested polymerase chain reaction assay for detection of Brucella DNA

ObjectiveTo establish a nested polymerase chain reaction (PCR) assay with high sensitivity and specificity to detect Brucella DNA.

MethodsThe bacteria DNA was extracted by using bacterial genome extraction kit. The DNA extraction from blood samples was conducted by using blood and other tissue genomic DNA extraction kit. The extracted DNA was pre-amplified by conventional PCR. The amplified PCR product was used again as the template for the second amplification by fluorescence quantitative PCR (nested PCR). The sensitivity and specificity for bacteria DNA extraction were tested. The relationship curve between Ct value and copy numbers of DNA was constructed. The DNA from blood samples was tested. The results were compared with conventional PCR and conventional fluorescence quantitative PCR.

ResultsThe sensitivity of conventional PCR was 512 copies of Brucella DNA. The effective range of genomic DNA was 921.6 ng/μl–6.8 fg/μl for nested PCR. And the corresponding Ct value was 12.04–37.50. The index relationship was y＝（e^−0.695x）×10¹², R²＝0.998 6. The amplification efficiency of nested PCR was 2.28×10⁹. The detection limit for nested PCR was 2 copy numbers of Brucella DNA. The sensitivity, specificity, positive predictive value and negative predictive value of nested PCR were 91.67%, 93.10%, 91.67% and 93.10%, respectively. Twenty five blood samples from a sheep farm were detected by nested PCR. The positive rate was 92.00% (23/25). The detection results of Brucella DNA were negative for 27 blood samples from healthy people (no Ct value).

ConclusionThe nested PCR has high sensitivity and specificity, which is suitable to use for the detection of Brucella in blood samples.