BF cirrhosae (bulbus of F. cirrhosa D. Don, F. unibracteata Hsiao et K. C. Hsia, F. delavayi Franch., F. taipaiensis P. Y. Li, F. przewalskii Maxim. and F. unibracteata Hsiao et K. C. Hsia var. wabuensis (S. Y. Tanget S. C. Yue) Z. D. Liu, S. Wang et S. C. Chen), BF ussuriensis (bulbus of F. ussuriensis Maxim.), BF pallidiflora (bulbus of F. pallidiflora Schrenk and F. walujewii Regel), BF thunbergii (bulbus of F. thunbergii Miq.) and BF hupehensis (bulbus of F. hupehensis Hsiao et K. C. Hsia) were gifted by Chengdu Institute Food and Drug Control and authenticated by DNA barcoding using ITS as a marker gene. The DNA of all BF samples was extracted using the plant DNA isolation Kit (Foregene Co., Ltd., Chengdu, China) according to the manufacturer’s instructions. The concentration of DNA was determined by the ultra-micro ultraviolet spectrophotometer ND5000 (BioTeke Co., Ltd., Beijing, China) and stored at 4°C. Finally, 1 μL DNA sample was directly added to the RALA system.

The total volume of the PCR reaction mixture was 20 μL, containing 1 × EasyTaq buffer (20 mM Tris–HCl (pH 8.4), 20 mM KCl, 2 mM MgSO₄, and 10 mM (NH₄)₂SO₄, 0.2 mM dNTPs, 3 U of EasyTaq polymerase, 0.2 μM each primer (Supplementary Table 1), and 1 μL DNA template. The PCR reaction mixture was placed in a C1000TM Thermal Cycler PCR (Bio-Rad Laboratories, United States) and denatured at 94°C for 2 min, followed by 35 thermal cycles (denaturation at 94°C for 5 s, renaturation at 56–60°C for 5 s, extension at 72°C for 20 s), and extension at 72°C for 2 min. The results were analyzed through 2% agarose gel and sequenced by BBI [Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China].

The RALA primers included a pair of inner primers and one (or two) loop primers were designed according to the specific site of the ITS sequence or the P16 gene sequence. All oligonucleotides were synthesized and purified by BBI, and the purification level was PAGE. The RALA primer sequences of 5 kinds BF were shown in Supplementary Table 2.

The total volume of the RALA reaction mixture was 25 μL, including 20 mM Tris–HCl (pH 8.8), 60 mM KCl, 4 mM Mg SO₄, 10 mM (NH₄)₂SO₄, 0.1% Triton X-100, 0.8 M betaine, 0.8 mM dNTPs, 1.6 μM inner primers (FIP and BIP), 0.8 μM loop primers (LF and LB), 0.24 U/μL Bsm polymerase, 0.6 mM ATP, 6 ng/μL TthRecA, 0.2 × SYBR Green I, and 1 μL DNA sample. Each group of primers amplified five BF at the same time. The reaction mixture was placed in the PikoReal Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, United States) at 60°C for 80 min. The results could be analyzed through the real-time collected fluorescence signals and 2% agarose gel.

Taking BF cirrhosae as the target, the analytical sensitivity of RALA was investigated by using a 10-fold serial dilution of pure genomic DNA (25 ng/μL –2.5 × 10^–5 ng/μL) and the mixed templates which contained different percentages of BF cirrhosae in the mixed genome with maize (100, 10, 1, 0.1, 0.01, and 0%, respectively, regards to total 25 ng/μL of genomic DNA).

N-red was used as the chromogenic reagent of RALA detection in the establishment of the colorimetric analysis platform for five BF. The colorimetric system is based on real-time RALA system, in which SGI is replaced by 200 μM N-red, and the reaction was incubated at 60°C for 80 min. The results of visual color reaction were captured with Canon 600D camera (Canon, Japan) under the background of white paper.

The genome sequence-based molecular detection leverages the specific gene fragment as the amplification target to accurately identify the medicinal plants of related species. After decades of practice, ITS sequence (China Plant BOL Group et al., 2011; Shi et al., 2020), rbcL sequence (Chase et al., 2005), and matK sequence (Hollingsworth et al., 2011) have been proved to be useful DNA barcodes for molecular identification of medicinal plants. Figure 1 illustrates the principle of the BF identification using our newly invented RALA. Firstly, the recombinase Tth RecA binds with the inner primers FIP and BIP to form the nucleoprotein complex and open the target double-strand DNA (dsDNA) at the consensus region. Next, the primers anneal to the complementary strand and are extended by the DNA polymerase with strand displacement ability. After the initial extension and strand displacement of the inner primer, the dumbbell-like amplicon is generated and self-primed for further extension. Moreover, the new inner primers can anneal to the dumbbell-like amplicon to induce the next round of extension. Besides, the loop primers LF and LB were designed to anneal to the single-strand loop domain of the intermediated products, which can further improve the amplification rate of RALA. The whole amplification process can be completed at a constant temperature of 60°C and the products of RALA amplification were considerable stem-loop DNAs with inverted target repeats. For different analysis needs, both the real-time fluorescence and colorimetric reporting system were established to present the results (which will be introduced in detail in the two chapters of Real-time RALA detection of BF cirrhosae and Colorimetric detection of BF cirrhosae).

Bulbus Fritillariae cirrhosae is the bulbus of six species of Fritillariae: (1) F. cirrhosa D. Don; (2) F. unibracteata Hsiao et K. C. Hsia; (3) F. przewalskii Maxim.; (4) F. delavayi Franch.; (5) F. taipaiensis P. Y. Li; (6) F. unibracteata Hsiao et K. C. Hsia var. wabuensis (S. Y. Tang et S. C. Yue) Z. D. Liu, S. Wang et S. C. Chen). Fortunately, the ITS sequences of the six source plants of BF cirrhosae are identical in large part, and there are multiple base sites different from the other BF (Supplementary Figure 1B). To develop the RALA-based detection method for BF cirrhosae, the ITS sequence region of the six kinds of BF cirrhosae was selected for primer design. Several sets of specific primers of BF cirrhosae were designed according to the alignment result of ITS sequences (Supplementary Table 3) and evaluated by amplifying the genomic DNA of various BF drugs (Supplementary Figure 2). As shown in Figure 2A, primer set BF cirrhosae-6 shows the highest specificity as the rising fluorescence curve only occurred when amplifying the genomic DNA of BF cirrhosae while the fluorescence curves remained flat when amplifying other BF genomic DNA, indicating no cross-reaction when amplifying non-target samples. The amplification products of Figure 2A were further verified by agarose gel electrophoresis. As depicted in Figure 2B, the typical band only appeared in amplification products of BF cirrhosae. These results demonstrated that the RALA-based assay could efficiently distinguish BF cirrhosae from their related kinds. It is worth mentioning that equal proportion mixed samples of six BF cirrhosae was used as BF cirrhosae samples for the described specificity detection as the sequence of the target fragment is almost identical among these six kinds of BF cirrhosae (Supplementary Figure 1). In order to verify it, the optimal primer set was used to detect all these six BF cirrhosae and the mixed samples, respectively. The rising fluorescence curves in Supplementary Figure 3 show that this primer set is able to detect all six kinds of BF cirrhosae and the mixed samples with a similar efficiency.

After the specificity examination, we sought to evaluate the sensitivity of this method. The serially diluted genomic templates of BF cirrhosae were used to determine the minimum detectable quantity of BF by RALA. As presented in Figure 3A, the reactions with template concentrations ranging from 25 ng to 2.5 pg manifested the rising fluorescence curves within 80 min, suggesting RALA could efficiently detect down to about 25 pg of the target template. To explore the quantitative ability of the real-time RALA assay, the Tq value defined as the reaction time when the fluorescence intensity exceeds the arbitrary threshold was introduced to the assay. As shown in Figure 3B, the Tq value linearly correlates with the target concentration (R² = 0.9702), indicating that this method has the potential to realize quantitative detection. To evaluate the sensitivity of the method in mixed samples, 25 ng of the mixed genome containing different ratios of BF cirrhosae genome (100, 10, 1, 0.1, 0.01, and 0%, in percentage) were used as the templates. Figure 3C shows that the amplification curve appeared even in detecting mixed templates containing 0.1% of the BF cirrhosae genome. The result was also verified by electrophoresis (Figure 3D), demonstrating that the RALA assay could detect trace amounts of BF cirrhosae in the mixed templates.

After the establishment of the real-time RALA assay for BF cirrhosae, we intended to extend its use to a simpler situation of field detection without the need for fluorescence monitoring. Many colorimetric dyes including calcein, phenol red, neutral red (N-red), and so on have been proved to be applicable in realizing on-site colorimetric isothermal detections (Tomita et al., 2008; Tanner et al., 2015). Among these dyes, N-red, with its distinct color changes from yellow to pink when the pH value decreases from 8.0 to 6.4, has been successfully applied in colorimetric LAMP assays (Ding et al., 2019; Wang et al., 2019). Since LAMP and RALA reactions have similar pH changes, we introduced the N-red to the RALA assay to establish colorimetric RALA detection for BF cirrhosae. The colorimetric detection workflow of BF is shown in Figure 4A. Firstly, DNA was extracted from fragmented BF samples by the kit column method, and then the RALA reaction with N-red was carried out. After incubation at 60°C for 80 min, the visual results were obtained. As shown in Figure 4B, the only tube containing the BF cirrhosae templates displayed evident pink color after the incubation. It could be easily discriminated from the yellow color of those tubes containing other non-target samples, suggesting that the N-red could be used in the RALA assay to reflect the amplification results. Then, we investigated the sensitivity of the colorimetric RALA assay. As illustrated in Figure 4C, the color of the tubes containing 25 ng to 2.5 pg of BF cirrhosae genome presented obvious pink, while the color of the rest tubes remained orange-yellow after the incubation, implying that the sensitivity of colorimetric RALA is 200 pg, which was consistent with the real-time assay (Figure 3A). And then we examined the sensitivity of the colorimetric detection method in mixed templates. As shown in Figure 4D, the colorimetric RALA assay could also distinguish as low as 0.1% BF cirrhosae from the mixed samples. It was worth noting that the N-red was preadded to the reaction tube, which could realize one-pot detection and decrease the carry-over contamination. Thus, the colorimetric RALA method could be an appealing choice for field detection of BF cirrhosae.

The successful establishment of RALA detection system for BF cirrhosae motivated us to develop the identification method for other common BF including BF ussuriensis (bulbus of F. ussuriensis Maxim.), BF pallidiflora (bulbus of F. pallidiflora Schrenk), BF hupehensis (bulbus of F. hupehensis Hsiao et K. C. Hsia) and BF thunbergii (bulbus of F. thunbergii Miq.). The ITS sequences of these four plants were aligned and the specific primer sets for BF ussuriensis and BF pallidiflora were designed (Supplementary Table 2 and Supplementary Figure 1). Due to the high similarity of ITS sequences between BF thunbergii and BF hupehensis, the ITS sequence could not distinguish the two with RALA reaction. Therefore, the RAPD marker p16 genomic sequence (Li et al., 2014; KF934438.1) of BF thunbergii was selected as the candidate target. We designed PCR primers of the P16 gene (Supplementary Table 1) to detect eight BF samples. As shown in Supplementary Figure 4, only BF thunbergii and BF hupehensis were amplified, and their products were sequenced and uploaded to the GenBank (the accession number were ON493814 and ON493815). By comparing the sequencing results, we obtained their specific sites and designed the RALA primers of BF thunbergii and BF hupehensis (Supplementary Figures 5 and 6). The specificity of these primer sets was parallelly determined by detecting all five kinds of BF independently. As shown in Figure 5, each primer set for BF hupehensis, BF ussuriensis, BF pallidiflora, and BF thunbergii showed a positive amplification curve only when amplifying their corresponding templates, indicating the high specificity of these primer sets. Similarly, one-pot colorimetric detection was also evaluated. As shown in Figure 6, the positive amplification tubes, which contained the specific primer set and its corresponding template, presented a color change from yellow to pink, while the amplification tubes containing the unmatched primer-template remained yellow. Therefore, these results demonstrated that the RALA-based identification system is successfully developed to detect all these marketable different BF, providing a universal tool to distinguish different kinds of BF.