Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology

Abstract

On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

Comparison of Influenza Virus Detection Methods

“…Recently, it has been reported that a microfluidic RT-PCR device yielded a similar sensitivity to the conventional thermal cycler RT-PCR device, where the time required for the amplification was approximately one-tenth of that of the conventional thermal cycler RT-PCR. (15,16) The sensitivity of the RT-PCR is higher than that of the plaque assay, because the RT-PCR detects RNA not only in infectious viruses but also in noninfectious ones. The sensitivity of the RT-PCR is considered to be sufficient to detect viruses in the environment and the RT-PCR is applied as a sensor for the prevention of infections.…”

Evaluation of the basic assay performance of the GeneSoc® rapid PCR testing system for detection of severe acute respiratory syndrome coronavirus 2

“…The system is expandable to run up to four detection units on a single main unit. All components of the system are integrally controlled [5,6]. The reaction kit and polymerase used for this study were the One Step Prime Script RT-PCR kit (Takara Bio Inc., Shiga, Japan) and Speed STAR HS DNA polymerase (Takara Bio Inc.), as described in Table 1A and 1B.…”

“…Recently, the innovative PCR system GeneSoC 1 (Kyorin Pharmaceutical Co. Ltd., Tokyo, Japan) that detects specific SARS--COV-2 RNA sequences using a microfluidic thermal cycling technology, has been developed. This system is capable of detecting a sequence specific to microbial pathogens in a sample within 10-15 min and can be operated on-site in a clinical setting [5,6].…”

Facile Fabrication of Thin-Bottom Round-Well Plates Using the Deformation of PDMS Molds and Their Application for Single-Cell PCR

“…Currently, resin products finer and more precise than conventional ones are contributing to the development of fundamental life science research and pharmaceutical and medical applications, such as cell analysis devices. For example, these so-called microdevices, such as microwell arrays for single-cell manipulation [ 4 , 5 ], microfluidic channels for rapid and high-throughput polymerase chain reaction (PCR) [ 6 , 7 ], and drug testing [ 8 , 9 ], and point-of-care testing (POCT) devices for blood cell analysis [ 10 ], have been studied extensively. Some of these are already commercially available as high-value-added life science/medical products for cell analysis [ 11 , 12 ].…”