Objective      To establish and evaluate a real-time PCR for fast

accurate and quantitative detection of Burkholderia pseudomallei (B. pseudomallei).       Methods      Based on B. pseudomallei type Ⅲ secretion system conservative sequence

primers and probe were designed

and then recombinant plasma was constructed as DNA standard. The real-time PCR system was optimized

and compared with other real-time PCR with 16S rRNA as target gene.       Results      The inventive real-time PCR showed wide range of linearity (1×10

1

-1×10

10

copies/μL)

high sensitivity (10 copies/μL)

and intra-experimental coefficients of variation (CV) of 1.43%-4.6%. When it was applied directly to identify 24 mocked double-blind blood samples

16 samples were positive to B. pseudomallei

and 8 samples were negative to other pathogens. Then

the inventive real-time PCR was further applied to identify 67 soil samples that underwent parallel bacteriological culture

and 12 soil samples showed B. pseudomallei positive results. There were 2 soil samples with positive results based on direct culture and the inventive real-time PCR

but other real-time PCR detection results were negative.       Conclusion       The inventive real-time RCR is rapid

accurate and sensitive for quantitative detection of B. pseudomallei

and can be used for diagnosis of melioidosis in epidemic area.