Primer generation-rolling circle amplification method optimized for the detection of pathogenic bacteria

• Published in: Biotechnology and bioprocess engineering Vol. 29; no. 5; pp. 890 - 901
• Main Authors: Jang, Eun-Jin, Kim, Tai-Yong, Lim, Jeong-A., Woo, Min-Ah
• Format: Journal Article
• Language: English
• Published: Seoul The Korean Society for Biotechnology and Bioengineering 01.10.2024; Springer Nature B.V; 한국생물공학회
• Subjects: Antisense DNA; Bacillus cereus; Bacteria; Biotechnology; Chemistry; Chemistry and Materials Science; Deoxyribonucleic acid; DNA; DNA polymerase; DNA probes; DNA-directed DNA polymerase; E coli; Efficiency; Enzymes; Escherichia coli; Escherichia coli O157; Food; genes; Industrial and Production Engineering; Listeria; Listeria monocytogenes; Methods; Microorganisms; Probes; Research Paper; Salmonella; Salmonella Typhimurium; Target detection; Thermal cycling; 생물공학; Bacteria; Rolling circle amplification; Detection; Isothermal amplification; Genomic DNA
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-024-00117-2

With advancements in DNA amplification research, isothermal amplification technology has emerged as an attractive method for detecting target DNA. Here, we describe primer generation-rolling circle amplification (PG-RCA) as an isothermal amplification method for detecting Escherichia coli O157:H7, Salmonella Typhimurium, Bacillus cereus , and Listeria monocytogenes . To improve PG-RCA sensitivity, the concentrations of the reaction components, dNTPs, phi29 DNA polymerase, and circular probes were optimized; the optimized conditions were applied to detect each target bacterium. A pair of forward and reverse circular probes that hybridized to the sense and anti-sense target genes was used in PG-RCA, exhibiting target selectivity. PG-RCA, which generated additional primers simultaneously with linear RCA and comprised multiple reaction cycles, resulted in higher accumulation of amplified DNA products than did linear RCA within the same reaction period. The threshold time (Tt) for each target gene concentration was determined based on the threshold value set in the amplification plot for PG-RCA, and a linear correlation between the Tt value and genomic DNA concentration was proven for each of the four bacteria. The PG-RCA-based assay could be applied to gene-based detection of various microorganisms and may be a useful isothermal amplification method for replacing traditional PCR methods.