Human cultivation and uses of Morinda officinalis How (MO) date to ancient Chinese, Japanese, and Indian cultures and now is widely spread in tropical, subtropical and temperate regions of Asia 1. Belonging to the family Rubiaceae, tasted sweet root with special brown color, MO has biological functions on liver, kidney, bone and muscle 2. It is also known to be beneficial to osteoporosis, rheumatoid arthritis, menstrual disorders, diabetes, rheumatoid and Alzheimer disease 3,4. More than 400 active chemical compounds have been isolated from this plant, and the major constituents have been found to be anthraquinones, iridoid glycosides, saccharides, fatty acids and triterpenoids 5. Crude extracts and pure compounds of the plant are used as effective agents in the treatment of many human biological diseases. In the market, many types of plants materials that resemble MO in appearance have been used to imitate MO because of unsustainable cultivation, over-harvesting, or economic reasons 6. Although the morphological characteristics of these imitations are similar to high-quality MO, they are cheaper and their pharmacodynamic effects are remarkably different. According to Chinese Pharmacopoeia (2015 edition), currently methods used to identify MO include macroscopic characterization, phytochemical methods 7 or molecular level analysis 8, such as TLC(Thin-Layer Chromatography), genetic markers9 and UHPLC-MS/MS10. These technologies regarded as benchmark assay because of authoritative, sensitive and stable, but would spend long run time with professional operation personnel and corresponding analysis infrastructure. It is very hard to identify them by the classical identification methods for consumers to immediately distinguish Morinda species, then causing confusion in market of medicinal materials. More seriously, MO adulterants obviously influence the clinical therapeutic effects and cause serious side effects 11. Therefore, a useful, fast and accuracy assay method for screening adulterated MO is urgently required.
The loop-mediated isothermal amplification (LAMP) method, established by Notomi in 2000, is a simple nucleic acid amplification technology with high efficiency and specificity 12. The whole work is able to completed in 40 ~ 60 min, with similar test limit to PCR (Polymerase chain reaction) technology13. Furthermore, compared with PCR-based methods, LAMP is several orders of magnitude more sensitive that can overcome the specificity limitations 14. The method requires 4 ~ 6 suitable primers that are specifically designed for recognizing and able to bind 6 ~ 8 precise gene regions, the whole process can be performed at 60 ~ 65 °C and only required bacillus stearothermophilus DNA (Bst DNA) polymerase 15. Figure 1 describes the DNA loop amplification process in detail. The amplification obtained single-stranded DNA by Bst DNA to achieve polymerase-mediated strand-displacement synthesis, thus obviating the step of thermal denaturation and the thermal cycler equipment 16. Since temperature changes are not required, the LAMP technology are promising approaches that allows the reaction to carry out applicably in a shorter time 17. Various types of DNA dyes can use directly and then observe with the naked eye without opening tubes because of large output of the amplification products 18. To avoid potentially subjective, relatively simple quantitative detection methods (turbidity or fluorescence), can revealed the results because of the high specificity. Additionally, there is no need for complicated operation and specific laboratory with highly sophisticated instrumentation during application, such as a thermal cycler and other imaging devices. Based on its rapidity, simplicity, and high specificity, LAMP assay give satisfactory results in widely fields, for example pathogen and genetic detection in clinical diagnosis such as global pandemic COVID-1919, fast microbe and harmful insect detection in agriculture products and environmental samples 20,21. However, authentication of MO from its adulterants by LAMP has not yet been established.
DNA barcode technology is an emerging technology that uses a conserved segment of biological DNA to quickly and accurately identify species 22. DNA barcodes is an efficacious molecular tool that can provide species-level resolution to distinguish various creatures differentiation, currently has been widely used in plant identification 23. The ITS2 sequence has been recommended as an effective core molecular marker of the DNA barcode 24, which can be used to verify a wider range of herbal species groups with high variability and species discrimination 25. These advantages make ITS2 sequence becomes a prospective barcode gene target to increase the success rate of LAMP assay. This study sought to establish a rapid and reliable LAMP assay method to distinguish MO from its adulterants based on the ITS2 sequence.