Species authentication/discrimination is an essential task in various areas in biology systematics, ecology, evolution, forensics, food science, medical as well as even herbal and cosmetic industries, leading to correct species exploitation regarding their purposes1–8. Traditional species taxonomy has been performed using the external morphological features or microanatomy which tightly requires the complete flower features or complete significant characteristics for species identification by an expert9. Perhaps, in several areas, the specimens obtained have been incomplete forms, immature stage, or modified/processed samples without key characters to identify, contributing to difficulty/impossibility in species identification and hampering the advance of investigation or research9. In several decades ago, advanced molecular approaches e.g., hybridization, DNA fingerprint, DNA barcodes, high resolution melting (HRM) have been used widely and extensively for facilitating species authentication in various organisms2–6, 10–16. Certainly, these molecular approaches enable species identification despite the specimens with completely damaged but DNA existing, especially DNA barcodes (Bar) which there are many regions exhibiting a successful species discrimination for plant species (e.g., rbcL, matK, trnL, and ITS)15–16. However, they are relatively complex, time and cost-consuming because they require expensive equipment (e.g., thermal cycler, realtime PCR, sequencer machine).
Currently, nucleic acid isothermal amplification (e.g., RCA, LAMP and RPA) has been emerging and gaining attention for RNA/DNA amplification, in particular pathogen detection as they require only heat box or water bath, leading to adaptation for point-of-care testing17–19. RPA is one of isothermal amplification based on enzymatic activities relating to DNA replication process and the reaction can be performed at constant temperature in range of 30–45°C for DNA amplification (optimal temperature at 37°C)18–19, mycoplasma20, and virus/viroid RNA in plant21.
Recently, CRISPR-cas systems exhibited the high potential for genome editing with accuracy and precise in the specific DNA target and included the adaptation for pathogen diagnostic with high sensitivity, specificity, simplicity, and speed, for instances, HPV-16 and 1822–23 and shrimp pathogens e.g., white spot syndrome virus (WSSV)24. The cas12a can be applied as diagnostic tool because it has the collateral activity or trans-activity for cleavage of non-target single stranded (ss) DNA once forming a tertiary complex (cas12a-gRNA-target)25. ssDNA is designed as reporter based on fluorescence resonance energy transfer (FRET) between fluorescence and its quencher or antigen-antibody interaction by lateral flow dipstick readout22–24, 26.
Herein, we would like to establish a novel method for plant species authentication using the hyphenation of plant DNA barcode, trnL and cas12a, namely “Bar-cas12a”. In this study, Phyllanthus species including Phyllanthus amarus, Phyllanthus urinaria, Phyllanthus debilis, Phyllanthus virgatus, were used as a model to validate Bar-cas12a for species authentication of P. amarus because they have similar morphological features and have been used as herbal commercialized products.