Abstract
Antimicrobial resistance, a global health concern, has been increasing due to inappropriate use of antibacterial agents. To facilitate early treatment of sepsis, rapid bacterial identification is imperative to determine appropriate antibacterial agent for better therapeutic outcomes. In this study, we developed a rapid PCR method, rapid cycle sequencing, and microchip electrophoresis, which are the three elemental technologies for DNA sequencing based on the Sanger sequencing method, for bacterial identification. We achieved PCR amplification within 13 min and cycle sequencing within 14 min using a rapid thermal cycle system applying microfluidic technology. Furthermore, DNA analysis was completed in 14 min by constructing an algorithm for analyzing and performing microchip electrophoresis. Thus, the three elemental Sanger-based DNA sequencing steps were accomplished within 41 min. Development of a rapid purification process subsequent to PCR and cycle sequence using a microchip would help realize the identification of causative bacterial agents within one hour, and facilitate early treatment of sepsis.
Highlights
The recent rise in antimicrobial-resistant bacteria due to improper use of antibacterial agents has become a global public health problem
By combining rapid polymerase chain reaction (PCR) technology with microchip electrophoresis technology, we achieved rapid identification of bacteria using rapid DNA sequencing based on the Sanger method, which is more reliable for DNA sequence analysis
We examined the rapid PCR conditions for 16S ribosomal DNA (rDNA) amplification using GeneSoC, which is a rapid real-time PCR system
Summary
The recent rise in antimicrobial-resistant bacteria due to improper use of antibacterial agents has become a global public health problem. MS) has recently attracted attention for rapid identification of bacteria, it can be used only for blood culture-positive samples due to the low limit of detection required to detect over 10,000 bacteria In both methods, broad spectrum antibacterial agents such as carbapenem are often used before the causative bacteria is identified, which increases the risk of antibacterial resistance developing. Nanopore-based sequencing is fast (10–180 min), identification of bacteria within one hour, which is important for early treatment of sepsis, has not been achieved since 16S rDNA PCR amplification takes a long time. By combining rapid PCR technology with microchip electrophoresis technology, we achieved rapid identification of bacteria using rapid DNA sequencing based on the Sanger method, which is more reliable for DNA sequence analysis.
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