, whereas the 162- and 147-bp mpr and zmp products were amplified from B. pseudomallei and B. cepacia, respectively (Fig.
1). All 66 B. pseudomallei, one B. thailandensis and four B. cepacia clinical isolates were positive for the groEL gene, indicating successful detection of the genus Burkholderia. All 65 B. pseudomallei isolates and K96243 strain were positive for the detection of mprA gene. Similarly, all three B. cepacia isolates and ATCC 25416 strain were positive for zmpA gene. Sequence analysis of the PCR products selleck from the amplification of groEL, mprA and zmpA matched the published gene sequences in the NCBI website. The negative control strains did not yield any PCR product, suggesting that the primers were highly specific for the different Burkholderia spp. In addition, no cross-reactions were observed within the Burkholderia spp. The mprA and zmpA genes were correctly amplified in the targeted strains, indicating
a specificity of 100%. Selleck RG7204 The limit of detection assay demonstrated that the groEL and zmpA PCR assay was sensitive at 10 pg mL−1 DNA, whereas mprA PCR assay was sensitive at 10 fg mL−1 (Figs 2 and 3). The PCR assay using DNA obtained from blood samples revealed successful amplification of B. pseudomallei in two of the 18 samples tested. On comparison with culture and API 20 NE results, these two PCR-positive samples were also positive for B. pseudomallei by culture and API 20 NE. The PCR-negative samples were also negative on culture, indicating sensitivity and specificity of 100%. However, none of the serum samples produced positive amplicons for any of the three primer sets. Duplex real-time PCR using SYBR green was performed using mprA (162 bp) and zmpA based on the melting curve analysis of amplified products. These primers allowed the amplification of PCR products with distinct melting temperature values, resulting buy Y-27632 in the formation of two distinct peaks
representing the two targets. The 167-bp amplicon of mprA (Tm 84 °C) could be clearly separated from the 147-bp amplicon of zmpA (Tm 88 °C) (Figs 4 and 5). No primer dimers were observed in the amplified product, which indicates the specificity of the primers. In this study, a conventional PCR assay was developed for the detection of Burkholderia genus and also for differentiation of the two clinically important human pathogens, B. pseudomallei and B. cepacia. Using bioinformatics tools, this assay incorporated detection of groEL gene, specific for the genus Burkholderia, mprA gene, specific for B. pseudomallei, and zmpA genes specific for B. cepacia. The groEL gene encodes an immunogenic protein of Burkholderia that assists in a proper protein-folding mechanism (Woo et al., 2001). blast analysis revealed that groEL is present in B. mallei, B. pseudomallei, B. cepacia, Burkholderia vietnamiensis and B. thailandensis among the Burkholderiaceae. Moreover, this gene sequence is highly conserved among all Burkholderia spp.