The principle of the SOE PCR assay

Four major drug-resistance-related gene fragments, i.e., rpoB, embB, katG, and the inhA promoter, were ligated and amplified into a fusion fragment of approximately 700 base pairs (bp) in a single reaction tube, after which the four drug-resistance-related gene fragments were sequenced via Sanger sequencing in one read, greatly accelerating the detection of the presence of drug-resistance mutations that may exist in a specific M. tuberculosis isolate.

There were three different overlapping complementary sequences between the primers rpoB_Rev (1R) and embB_For (2F), between primers embB_Rev (2R) and katG_For (3F), and between primers katG_Rev (3R) and inhA_For (4F) respectively, as indicated in different colors in Fig. 1. In the SOE PCR reaction system, the concentrations of the primers 1F and 4R is higher than that of the other six primers, while the concentrations of the primers 2R and 3F is higher than that of the primers 1R, 2F, 3R, and 4F.

Schematic diagram of the principle of SOE PCR. SOE PCR: Gene splicing by overlap extension PCR

At the initial stage of the SOE PCR reaction, double-stranded DNA fragments of rpoB, embB, katG, and inhA were produced. As the reaction cycles increase, the primers 1R, 2F, 3R, 4F are depleted and exhausted, and the single-stranded DNA fragments of rpoB, embB, katG, and inhA are produced. During this process, there is a probability of gene splicing between the fragments rpoB and embB, and between the fragments katG and inhA.

Then, as the reaction cycles increase, the primers 2R and 3F are also depleted and exhausted, and the single-stranded DNA fragments of spliced rpoB-embB and spliced katG-inhA are produced. During this process, there is a probability of gene splicing between the spliced rpoB-embB and the spliced katG-inhA.

Once a small amount of the rpoB-embB-katG-inhA fusion fragment is produced in the SOE PCR system, it can then be used as a template to be amplified by primers 1F and 4R at both ends. The principle of SOE PCR in this study is illustrated in Fig. 1.