title: One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV.
abstract: Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 UBstpolymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices.
authors.affiliation: School of Biological Sciences, Chungbuk National University Cheongju, South Korea.
authors.forename: Se Hee
authors.initials: SH
authors.lastname: Lee
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University Cheongju, South Korea.
authors.forename: Yun Hee
authors.initials: YH
authors.lastname: Baek
authors.affiliation: School of Biological Sciences, Chungbuk National University Cheongju, South Korea.
authors.forename: Yang-Hoon
authors.initials: YH
authors.lastname: Kim
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University Cheongju, South Korea.
authors.forename: Young-Ki
authors.initials: YK
authors.lastname: Choi
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University Cheongju, South Korea.
authors.forename: Min-Suk
authors.initials: MS
authors.lastname: Song
authors.affiliation: School of Biological Sciences, Chungbuk National University Cheongju, South Korea.
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Ahn
keywords: EvaGreen; MERS-CoV; loop-mediated isothermal amplification (LAMP); nucleocapsid (N) protein; one-pot reverse transcription (RT)