Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.
As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amount of DNA is synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combines with divalent metallic ion to form an insoluble salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction solution allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30-60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.
Tomita
Norihiro
N
Eiken Chemical Co., Ltd., Biochemical Research Laboratory, 1381-3 Shimoishigami, Ohtawara, Tochigi 324-0036, Japan. norihiro_tomita@eiken.co.jp
Mori
Yasuyoshi
Y
Kanda
Hidetoshi
H
Notomi
Tsugunori
T
D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000235:genetics; D011756:Diphosphates / Q000737:chemistry; D005452:Fluoresceins; D023281:Genomics / Q000379:methods*; D006801:Humans; D008345:Manganese; D008670:Metals / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*
D017931:DNA Primers; D011756:Diphosphates; D005452:Fluoresceins; D008670:Metals; D008345:Manganese; D004247:DNA; C007740:fluorexon