title: Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay.
abstract: This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn2+dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25-45 bp, beacon concentration of 0.6-1 pmol/μL, and reaction temperature of 60-65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product.
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Simo
authors.initials: S
authors.lastname: Huang
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Ningwei
authors.initials: N
authors.lastname: Liu
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Derong
authors.initials: D
authors.lastname: Dong
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Zhan
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Ma
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: He
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Da
authors.initials: D
authors.lastname: Ao
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Yaqing
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Dayang
authors.initials: D
authors.lastname: Zou
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Liuyu
authors.initials: L
authors.lastname: Huang