Optimization of ultra-fast convection polymerase chain reaction conditions for pathogen detection with nucleic acid lateral flow immunoassay

서지정보

ㆍ발행기관 : 대한구강생물학회 ㆍ수록지정보 : International Journal of Oral Biology / 44권 / 1호
ㆍ저자명 : Tae-Hoon Kim, Hyun Jin Hwang, Jeong Hee Kim

목차

Introduction
Materials and Methods
1. Bacteria strains and preparation of genomic DNA
2. Convection polymerase chain reaction
3. Detection of polymerase chain reaction amplification products by electrophoresis and nucleic acid lateral flow analysis
Results and Discussion
1. Optimization of convection polymerase chain reaction conditions for single line nucleic acid detection
2. Optimization of conditions for double line nucleic acid detection
References

영어 초록

Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to 3 μM or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, 2 μM of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.

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