The PCR reactions were carried out in a final volume of 50 μL containing 1 × PCR buffer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 μM of each primer, 1.25 U of Trichostatin A Taq DNA Polymerase and 5 μL of DNA template and distilled water. Initial denaturation was performed at 94 °C
for 5 min, followed by amplification comprising 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s and extension at 72 °C for 45 s. A further 2-min final extension at 72 °C was carried out following the final cycle. The amplified PCR products were analyzed using 1.5% agarose gel (Promega) electrophoresis in 1 × TBE buffer at 90 V for 1 h and visualized using ethidium bromide staining under UV illumination. The positive PCR products were purified using Wizard PCR Purification Kit (Promega) and confirmed by sequencing (Research Biolabs
Sdn. Bhd, Singapore). The limit of dilution was determined by subjecting the DNA of the targeted organisms to PCR after 10-fold serial dilutions to produce a DNA concentration ranging from 10 μg mL−1 to 10 fg mL−1. Real-time duplex PCR amplification and melt curve analysis were carried out in an iQ5 real-time PCR detection system (BioRad Laboratories, Hercules, CA). QuantiTect SYBR green PCR SCH727965 order kit (Qiagen) was used for amplification with 0.3 μM of mprA and 0.2 μM of zmpA primers. The PCR was performed with the following cycling protocol. Initial denaturation for 15 min at 95 °C was followed by 30 cycles with 15 s at 94 °C, 30 s at 52 °C and 30 s at 72 °C. Fluorescence data were captured at the elongation step of each cycle. Following amplification, melt curves were acquired by increasing the temperature from 65 to 95 °C at the rate of 0.5 °C 10 s−1, with continuous measurement Methamphetamine of the fluorescence. In general, all three query gene sequences retrieved from the GenBank
and analyzed by blast were correct with an exact match of 100% identity. clustalw alignment revealed that the groEL gene sequence of B. pseudomallei was highly homologous to B. mallei, B. thailandensis and B. cepacia, with a score of 99%, 97% and 95%, respectively. The alignment scores of other organisms such as the Pseudomonads, Xanthomonas campestris, Bordetella pertussis and Ralstonia picketti displayed a distant relation to Burkholderia spp. Therefore, the regions of groEL appropriate for primer design were targeted at the part where there was 100% identity of bases among Burkholderia spp. and vast variation with other organisms. The mprA gene sequenced was not aligned with any other organisms as no database was found for a similar gene in other organisms. The zmpA of B. cepacia was aligned with that of B. pseudomallei. Alignment results revealed an identity of 86% between these two sequences. Thus, the regions that displayed significant nucleotide variation within zmpA sequences of these two organisms were chosen for primer design.