Rapid and Sensitive Detection of Lophophora williamsii by Loop-Mediated Isothermal Amplification

Abstract

In plants, nucleotide alignment of specific DNA regions, such as nuclear ribosomal RNA or chloroplast DNA, has been used for the determination of phylogenetic relationships and species classification.1-3) Such DNA information has also been used to authenticate herbal medicine in pharmacognosy. 4,5) There have been attempts to use DNA polymorphism-based detection techniques-commonly employed in the life sciences-for the authentication of medicinal plants. Such techniques include amplification-refractory mutation system (ARMS), 6) polymerase chain reaction (PCR)-restriction fragment-length polymorphism (PCR-RFLP), 7) and TaqMan assay.8) However, not all techniques can be applied directly to plant materials because total DNA from plants contain many secondary metabolites and polysaccharides. In PCR amplification, difficulties are sometimes encountered when total DNA extracted from plant materials was used as template without preliminary preparation of the sample via purification or technical modification.9,10) Moreover, the sequence selectivity of oligonucleotides such as PCR primers is decreased in reaction solutions that contain impurities. As many herbal medicines are derived from underground parts having high polysaccharide content, we saw the need to develop a new method to overcome this problem.Loop-mediated isothermal amplification (LAMP) technique has been developed and used in the detection of microorganisms. [11][12][13] As LAMP employs 4 to 6 primers, it involves 4 to 6 reactive sites and 6 to 8 recognition regions, giving it increased sensitivity and specificity compared to typical PCR. Moreover, the isothermal reaction condition increases time efficiency compared to PCR, which requires three temperature steps. LAMP achieves detection in one to two hours. We have already observed that LAMP has good amplification ability in the detection and identification of Curcuma plants.14) LAMP was successful even though Curcuma rhizomes have high polysaccharide content, which made extraction of total DNA difficult.The genus Lophophora, Cactaceae, is distributed in Central and South America; L. williamsii, L. diffusa, and several other varieties are included in this genus. 20) In this manuscript, we have adopted the theory that these two species are different. L. williamsii is known by its common name, peyote. Because it contains mescaline, a psychedelic, its use is regulated in some countries. There should be the method to indentify this species but there is not because it is very difficult to differentiate those two species morphologically. The species-specific detection methods are all the more nonexistent. Thus, we have analyzed the nucleotide alignment of the chloroplast trnL intron region in L. williamsii and L. diffusa and clarified the differences. Three sequence patterns were observed in L. williamsii (DNA Data Bank of Japan (DDBJ) accession Nos. AB362488, AB362489, and AB362490), and one pattern was observed in L. diffusa (AB362491). We found that these two species have different sequences in the trnL int...

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.…”

Rapid and Sensitive Identification of the Herbal Tea Ingredient Taraxacum formosanum Using Loop-Mediated Isothermal Amplification

“…Recently, a number of studies have demonstrated that LAMP can be applied to the detection of various plants, including genetically modified organisms (GMOs) [ 11 , 12 , 13 ]. However, LAMP has yet to be used to authenticate medicinal herbs [ 14 , 15 , 16 ]. LAMP is time-effective, with high specificity and sensitivity and requires relatively simple equipment; consequently, it has become a very powerful tool for developing a nucleic acid-based diagnostic method for identification or authentication of bio-resources.…”

Single Nucleotide Polymorphism Assay for Genetic Identification of Lophophora williamsii*

“…Lophophora williamsii identification using sequencing chloroplast DNA region such as rbcL and trnL-trnF IGS, which contains 400-900 base pairs (bp), has also been reported (11,12). A previous study reported the application of loop-mediated isothermal amplification (LAMP) using six specific primers, including two loop primers, to evaluate L. williamsii and L. diffusa DNA by measuring turbidity due to the formation of magnesium pyrophosphate in real-time monitoring of LAMP (7). The LAMP method facilitates fast amplification speed and high specificity, although the experimental process is complex and interpreting the results requires technical skills (13).…”

“…Lophophora diffusa , which does not contain mescaline, is similar in appearance to L. williamsii . Thus, L. williamsii or L. diffusa may be incorrectly identified when evaluating morphological features alone (7). In addition, there is concern that L. williamsii can be used for grinding, brewing, and powdered for capsules (8).…”