Rapid Identification of Curcuma longa and C. aromatica by LAMP

Abstract

We have developed a novel method for the identification of Curcuma longa and C. aromatica called "loop-mediated isothermal amplification (LAMP)," based on trnK gene sequences. LAMP employs four primers that recognize six regions on the target DNA. Cycling elongation was initiated when the four primers were annealed to the target DNA. Amplifications were detected by measuring turbidity due to the formation of magnesium pyrophosphate. We designed allele-specific primer sets for C. longa and C. aromatica, respectively. LAMP using a primer set for C. longa and total DNA extracted from C. longa rhizome (0.5-10.0 ng) as template was detected up to 70 min. On the other hand, in the reaction using a primer set for C. longa and total DNA from C. aromatica as template, no amplifications were detected. The same tendency could be seen in the reactions using a set of primers for C. aromatica. LAMP enabled not only identification but also detection with high specificity. This rapid, specific, sensitive, and convenient method is expected to be applicable to the identification of the botanical origin of commercially available herbal products.Key words Curcuma; trnK gene; loop-mediated isothermal amplification; identification; detection; Zingiberaceae Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies specific nucleotide sequences with high specificity, efficiency, and rapidity under isothermal conditions. 1) Compared to polymerase chain reaction (PCR), the four primers that recognize six nucleotide regions enable rapid amplification in LAMP. Amplifications are detected by electrophoresis or measurement of turbidity due to the formation of magnesium pyrophosphate.2) Because of its high specificity, LAMP has been used in the detection of microorganisms 3) or the genotyping of cytochrome P450 gene. 4)Recently, there is an abundance of health foods or supplements originating from plants in the market. However, a number of them are of inferior quality because they use materials that differ in botanical origin. As those products may have harmful effects, they should be banned from the market. The fact that most of them are difficult to authenticate indicates the need to devise a sensitive method for identification.Herbal products derived from Curcuma longa or C. aromatica rhizomes are often misused or are a mixture of the two plants because those two species are highly similar. We previously clarified differences in nucleotide sequences in nuclear 18S rRNA gene and plastid trnK gene among six Curcuma species distributed in China and Japan. 5) C. longa and C. aromatica were distinguished in terms of nucleotide alignment in the trnK gene. These species-specific regions made it possible to identify Curcuma plants and drugs by sequence analysis. In our previous study, we developed the amplification-refractory mutation system (ARMS) 6) and singlenucleotide polymorphism (SNP) analysis 7) for Curcuma species. In the present study, we used LAMP to identify Curcuma plants.One specimen eac...

Rapid Detection of Panax ginseng by Loop-Mediated Isothermal Amplification and Its Application to Authentication of Ginseng

“…In our previous work, amplification was detected from 55 to 70 min at DNA concentrations of 0.5 to 10.0 ng, which are the same as the concentrations used in this study. 4) Moreover, the set of primers for P. ginseng could be used to distinguish P. ginseng from P. japonicus despite the very slight differences in alignment. In genus Panax, P. japonicus is most closely related to P. ginseng genetically.…”

“…Many convenient methods to detect single-nucleotide polymorphisms (SNPs) have been developed, including polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP) 1) and TaqMan assay. 2) In particular, loop-mediated isothermal amplification (LAMP) 3,4) is convenient because the reaction could be conducted under isothermal conditions, thereby facilitating amplification. LAMP employs four kinds of primers and amplification proceeds only when all the primers are annealed to the target DNA.…”

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“…[160][161][162] In another study, Sasaki and Nagumo reported rapid identification of C. longa and C. aromatica by LAMP method. [163] A study conducted by Sigrist et al showed genetic diversity of turmeric germplasm (C. longa; Zingiberaceae) using microsatellite markers to discover genetics and molecular research.…”

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of <i>Cannabis sativa</i>

“…19,20) It does not need any special devices or experienced technicians; thus, it is widely used for various purposes, such as detecting viruses and medicinal herbs. [21][22][23][24] One of the most species-specific genes in C. sativa is the THCA synthase gene that consists of a 1632 bp open reading frame. THCA synthase gene was cloned and polymorphism analysis revealed that 63 nucleotide substitutions divided the species into two chemotypes: the THCA-rich type (drug type), such as marijuana, and the THCA-poor type (fiber type), such as hemp.…”