Normal PCR was conducted in a 20 μL reaction mixture containing 105 copies of template DNA, 250 μM dNTPs, 0.05 U/μL i-Taq DNA polymerase, 500 nM forward primer, and 500 nM reverse primer in 1X PCR reaction buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl, and 2 mM MgCl2). For PCR amplification, the reaction mixtures were heated to 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and finalized at 72 °C for 5 min23,24,35. Previously prepared Neisseria gonorrhoeae plasmid DNA (target DNA)47 and Leptospira interrogans genomic DNA (non-complementary (NC) DNA) were employed as template DNAs for amplification. To obtain 221-mer ssDNA, asymmetric PCR was conducted by following the same procedures but using different forward and reverse primer concentration ratios (5:1, 10:1, and 20:1) (Figure S8).

After completion of the reaction, a 5 μL aliquot of the PCR products was resolved on a 3% agarose gel containing EtBr, and electrophoresis was carried out at a constant voltage of 100 V for 40 min using 1X TBE as a running buffer. After gel electrophoresis, an image of the gel was taken with a Gel Doc EZ Imager (Bio-Rad, CA, USA). The PCR products were purified using a NucleoSpin Gel and PCR clean-up kit (Macherey–Nagel, Düren, Germany), and the concentrations of the purified products were measured using a Nanodrop One spectrophotometer (Thermo Scientific, MA, USA).

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