Alysis with all the certain probe, we performed a conventional HRM analysis working with DNA fragments of seven genotypes (AA, TA, GA, AG, CA, AC, and AT). The DNA fragment of your AA genotype was distinguished from DNA fragments of GA, AG, CA, and AC genotypes by HRM analysis at positions 2058 to 2059 on the 23S rRNA gene (Fig. S1). Even so, there were slight variations involving the DNA fragments of AA, TA, and AT genotypes. These benefits recommend that a traditional HRM assay alone can hardly discriminate involving the AA genotype and also other genotypes at positions 2058 to 2059 in the 23S rRNA gene.January/February 2023 Volume 11 Concern 1 ten.1128/spectrum.04326-22Rapid Screening Assay for Clarithromycin-Resistant MACMicrobiology SpectrumFIG 1 Partial sequence motif of 23S rRNA domain V from reference strains (Mycobacterium avium 104 and M.Isovitexin References intracellulare ATCC 13950). The particular probes applied within this study possess a genotype-specific sequence at NN (AA, TA, GA, AG, CA, AC, and AT).Lucigenin Fluorescent Dye Melting curve analysis of DNA fragments working with the AA-specific probe.PMID:32926338 At positions 2058 to 2059 of your 23S rRNA gene, the AA genotype MAC would be the sole strain susceptible to clarithromycin, and six other genotype strains are resistant to clarithromycin (19). Thus, we developed a screening assay to detect clarithromycin-resistant MAC using melting curve analysis with the AA-specific probe. Fig. two shows the melting peak plots of DNA fragments of seven genotypes by melting curve analysis applying the AA-specific probe. The melting peak plots derived from PCR amplicons were higher than 84 (Fig. 2C), similar to those inside a standard HRM assay. Because the 39 end in the AA-specific probe wasFIG two Melting peak plots on the DNA fragments of seven genotypes with the AA genotype-specific probe. Normalized melting peak plots (A) were acquired using DNA fragments in the AA (red line), TA (blue line), GA (orange line), AG (green line), CA (brown line), AC (purple line), and AT (gray line) genotypes. Each DNA fragment has two melting peaks derived from the probe (B) and the PCR amplicon (C).January/February 2023 Volume 11 Concern 1 ten.1128/spectrum.04326-22Rapid Screening Assay for Clarithromycin-Resistant MACMicrobiology SpectrumTABLE 1 Melting temperature values of the DNA fragments of seven genotypes with precise probesDNA fragment AA TA GA AG CA AC ATaTheTm for particular probea AA 80.33 0.ten 77.06 6 0.13 76.84 six 0.14 76.61 six 0.13 77.52 six 0.07 77.46 6 0.08 77.29 6 0.05 TA 77.33 6 0.31 79.76 0.23 77.02 six 0.33 75.27 6 0.28 77.92 6 0.22 76.19 6 0.28 76.23 six 0.03 GA 77.83 6 0.11 78.25 6 0.09 81.12 0.ten 76.99 six 0.19 78.00 6 0.11 76.58 six 0.18 76.22 six 0.09 AG 77.58 6 0.24 75.78 6 0.23 76.84 six 0.28 81.06 0.20 75.98 six 0.23 77.81 six 0.21 78.56 6 0.07 CA 77.49 six 0.03 77.00 six 0.05 77.34 6 0.03 75.60 six 0.04 81.77 0.06 77.88 6 0.07 75.61 6 0.02 AC 77.22 six 0.12 75.69 6 0.09 75.71 six 0.13 76.97 6 0.12 77.25 6 0.11 81.72 0.09 76.61 6 0.08 AT 77.24 6 0.07 76.29 6 0.06 75.56 six 0.07 76.73 6 0.10 75.92 six 0.08 78.02 six 0.13 79.75 0.melting temperature (Tm) values are expressed as imply six standard deviation of the triplicate independent assay. The highest Tm values among each probe assay are in boldface.phosphorylated, asymmetric PCR amplified the quick double-stranded DNA, which had a low melting peak beneath 82 (Fig. 2B). The melting temperature (Tm) value below 82 of each and every melting curve plot was automatically calculated applying Gene Scanning software program. The Tm worth from the DNA fragment in the AA genotype (80.33 6 0.1.
