title: Visualized detection of single-base difference in multiplexed loop-mediated isothermal amplification amplicons by invasive reaction coupled with oligonucleotide probe-modified gold nanoparticles.
abstract: Loop-mediated isothermal amplification (LAMP) is a well-developed DNA amplification method with an ultra-high sensitivity, but it is difficult to recognize a single-base difference (like genotyping) in target-specific amplicons by conventional detection ways, such as the intercalation of dyes into dsDNA amplicons or the increase of solution turbidity along with the polymerization process. To allow genotyping based on LAMP suitable for POCT (point-of-care testing) or on-site testing, here we proposed a highly specific and cost-effective method for detecting a single-base difference in LAMP amplicons. The method includes three key steps, sequence amplifier to amplify multiple fragments containing the single nucleotide polymorphisms (SNPs) of interest, allele identifier to recognize a targeted base in the amplicons by invasive reaction, and signal generator to yield signals by hybridization-induced assembly of oligonucleotide probe-modified gold nanoparticles. Because the allele identifier is sensitive to one base difference, it is possible to use multiplexed LAMP (mLAMP) to generate amplicon mixtures for multiple SNP typing. Genotyping of 3 different SNPs (CYP2C19*2, CYP2C19*3 and MDR1-C3435T) for guiding the dosage of clopidogrel is successfully carried out in a 3-plex LAMP on real clinical samples. As our method relies on the naked-eye detection and constant-temperature reaction, no expensive instrument is required for both target amplification and sequence identification, thus much suitable for inexpensive gene-guided personalized medicine in source-limited regions.
authors.affiliation: Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.
authors.forename: Xueping
authors.initials: X
authors.lastname: Ma
authors.affiliation: Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.
authors.forename: Jianping
authors.initials: J
authors.lastname: Wang
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Sheng
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Tianhui
authors.initials: T
authors.lastname: Dong
authors.affiliation: Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Qinxin
authors.initials: Q
authors.lastname: Song
authors.affiliation: Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Rui
authors.affiliation: Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China. Electronic address: zbj523@163.com.
authors.forename: Bingjie
authors.initials: B
authors.lastname: Zou
authors.affiliation: Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China. Electronic address: ghzhou@nju.edu.cn.
authors.forename: Guohua
authors.initials: G
authors.lastname: Zhou
mesh_terms: D015374:Biosensing Techniques; D065731:Cytochrome P-450 CYP2C19 / Q000235:genetics / Q000302:isolation & purification*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D005838:Genotype; D006046:Gold / Q000737:chemistry; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
chemical_list: D015345:Oligonucleotide Probes; D006046:Gold; D004247:DNA; D065731:Cytochrome P-450 CYP2C19
keywords: AuNPs; Invasive reaction; Visualized detection; mLAMP