Ex taq premix tli rnaseh plus

A multiplex protocol of Taq Man real-time PCR for the detection and differentiation between the MTBC members was performed to test the DNA samples extracted from EPTB human specimens, as well as positive and negative controls, following a protocol previously described by Halse et al [6 (link)]. We have used the same primers and probes of the original protocol for RD1, RD9, RD12 and ext-RD9 targets without the use of RD4 one since they are sufficient to distinguish between MTBC members as demonstrated in Table 1 [6 (link)]. Briefly, the qPCR was performed in a 25 μl final volume with Ex Taq Premix Tli RNaseH Plus (Takara, Japan) as described previously [6 (link)]. QPCR was performed on a CFX96TM real-time PCR cycler (Biorad, USA). Pure DNA was amplified in duplicate without and with a 1:5 dilution. Thorough preventive measures were taken to avoid DNA contamination during extraction and qPCR manipulation as mentioned previously [15 (link)].

The qPCR was run in a taqman mastermix containing 10 μl of Ex Taq Premix Tli RNaseH Plus (Takara, Japan), 250 nM of each invA primer, 100 nM of invA probe, and 2 μl of purified DNA in a final volume of 20 μl using nuclease-free water. Regarding the IAC, 320 nM IAC primers, 160 nM IAC probe, and 0.5 μl of IAC DNA template (0.5 pg μl^-1, approximately 10² to 10³ copies per PCR reaction) were added per reaction as described previously (Deer et al., 2010 (link)). CFX96TM real-time PCR thermocycler (Biorad, France) system was used for all the qPCR experiments. The optimal qPCR efficacy was achieved using cycling profile including a denaturation at 95°C for 30 s, followed by 40 cycles of 05 s at 95°C and 30 s at 60.0°C. Each qPCR included one positive control (S. enterica DNA), no template control (mastermix and sterile water) and a negative DNA control (Escherichia coli DNA). All qPCR experiments were performed in duplicate.