Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR)
Specific targeted alleles and PCR conditions for BeijingMycobacterium tuberculosis strain A (Beijing A) were as described elsewhere (Dominguez et al., 2019). In brief, PCR reaction mixtures included a final volume of 25 µl: 1.25 µl of 5 U/µl HotStarTaq® DNA polymerase (Qiagen, Hilden, Germany), 0.4 µl of 25 mM MgCl2, 2.5 µl of 10x PCR buffer with 15 mM MgCl2, 5 µl of 5x of Q solution, primer pool concentration (2.5 µl mix: 2µM, 6 µM, 5 µM), 0.2 µl of 25 mM dNTPs, water, and the DNA target. PCR conditions were as follows: 95 ºC for 15 min, followed by 27 cycles of 95 ºC for 1 min, annealing 64 ºC for 1 min, and 72 ºC for 10 min. The ASO-PCRs were performed on the isolates from September 2018 and July 2019 in Madrid (Spain) and the technique was transferred to Colon (Panama) for prospective in situ application on the new incident cases (August 2019 to March 2020).