Twistamp basic kit

In the two-step RPA-Cas13a assay, basic RPA reactions were first conducted according to the instructions of the TwistAmp Basic Kit (Twist Dx, Cambridge, United Kingdom). Each RPA reaction was carried out in a 50 μL reaction volume containing Primer Free Rehydration buffer 29.5 μL, 0.24–0.96 μM forward and reverse primers each 0.5 μL, target DNA template 2 μL, and RNase-free ddH₂O 15 μL. The reaction mixes were vortexed and spun briefly, and then 280 mM magnesium acetate (MgOAc) 2.5 μL was added and mixed well to start a reaction in fluorescence detector F1620 (Qitian, China) at 37, 39, and 41°C for 20 min. Then, a 25 μL CRISPR-Cas13a reaction system was performed with 5 × reaction buffer 5 μL, 25 mM NTP mix 1 μL, Recombinant RNase Inhibitor 0.5 μL, 1.2 μM Salmonella-crRNA 0.6 μL, 10 μM RNA-probe 0.5 μL, T7 RNA Polymerase 0.3 μL, 1 μM Cas13a 1 μL, and added RNase-free ddH₂O to 23 μL, and RPA products 2 μL. The fluorescent signals were collected at 39°C for 30 min in fluorescence detector F1620.

The species-specific sequences of internal transcribed spacers (IST) 1 and 2 between the 16S and 23S rRNA gene loci are used for identification of microbes at species and sub-species level. ITS1 and ITS2 from E. fawcettii were downloaded from NCBI and used as templates for the primer design, as previously reported [7 (link)]. Several species-specific candidates forward (F) and reverse (R) primer sets (Table 1) were manually designed according to the TwistAmp Manufacturer’s Principles Kit (TwistDX, Cambridge, UK). The primers were synthesized by Bioneer (Seoul, Korea).
The RPA reactions were established according to the instructions in the TwistAmp Basic Kit (TwistDX) with few modifications. Briefly, a mixture of 2 μL of template DNA, 1 μL of forward primer, 1 μL of reverse primer, and 29.5 μL of rehydration buffer (TwistDX) was finalized to 47.5 μL with double-distilled water, and 2.5 μL of magnesium acetate (MgOAc) was added before starting the reaction. The reactions were performed at 37 °C for 20 min.

RPA was performed using the TwistAmp^™ Basic kit (TwistDx, Cambridge, UK). Briefly, 12.5 μL of the total reaction volume contained the following: 1 × rehydration buffer, 480 nmol L⁻¹ of both forward and reverse primers, 2 μL diethylpyrocarbonate water, 2.5 μL of the DNA template and 1 μL of magnesium acetate (MgOAc; final concentration: 14 mmol L⁻¹). The reaction tubes were incubated at 37 °C for 15 min, including a manual mixing step (5-s tube vortex) in the fourth minute. For the no-template control (NTC), these reactions were prepared by substituting the DNA template with an equal volume of molecular grade water.
After RPA, 12.5 μL of RPA products were mixed with 7.5 μL of Cas12a reaction mixture, which contained 100 nmol L⁻¹ of crRNA, 50 nmol L⁻¹ of ssDNA reporter, 50 nmol L⁻¹ of LbaCas12a (EnGen LbaCas12a, M0653T, NEB), 1 × NEBuffer 2.0 (New England Biolabs, UK), and 1.5 μL of RNase-free water, for a final volume of 20 μL. Then, the reaction was incubated at 37 °C using the Applied Biosystems ^™ Quant Studio 3 (Thermo Fisher Scientific, USA) and fluorescence was measured every minute.

The detection of ASFV by probe-based qPCR has been described previously [29 (link)–31 (link)]. In terms of African swine fever virus (ASFV), qPCR is the gold standard, which we used as a reference for our CRISPR-Cas12a assay. LbCas12a (New England Biolabs) was used for this assay. As described in the DETECTR method, our CRISPR-Cas12a assay also uses recombinase polymerase amplification (RPA). For RPA reactions, the TwistAmp Basic kit (TwistDx) was used according to the manufacturer’s instructions (https://www.twistdx.co.uk/en). Briefly, 50 μl reactions containing 2.5 μl ASFV DNA, 0.48 μM forward and reverse primers, 1× rehydration buffer, 14 mM magnesium acetate and RPA mix were incubated at 39 °C for 20 min. CRISPR-Cas12a detection was performed as described previously with minor modifications. The reaction volume was a total of 50 μl, with 20 nM crRNA, 115 nM single-stranded DNA fluorophore quencher - labeled reporter (Table S1), 30 nM LbCas12a, 1 μl RPA amplification products, 2 μl RNase inhibitor (New England Biolabs), and 1 × LbCas12a Buffer (New England Biolabs). The reactions were incubated in a temperature-controlled water bath for 15 min at 37 °C. Fluorescence emission was excited at 485 nm and detected at 535 nm using a fluorescent microplate reader (BioTek), and reactions without target DNA were used to establish the background.

The sequences used for RPA primers design were obtained from the NCBI Nucleotide Database (GenBank accession numbers MH858319.1 and NR_131265.1). Primers for RPA were listed in Table 2. RPA reactions were performed by the Twist-Amp basic kit (TwistDX, British). Each RPA reaction (50 µL) contained 29.5 µL rehydration buffer, 2.4 μL forward and reverse primers, 2 µL genome DNA extraction samples, 2.5 μL of 280 mM magnesium acetate (MgAc), and 11.2 μL water. The mixtures were incubated at 39 °C for 15 min. For gel analysis, the RPA products were cleaned up using 70% ethanol precipitation method and verified by electrophoresis on a 1% agarose gel. For Cas12a detection, the 1 μL of RPA products without purification were directly added to Cas12a reaction. For the limit of detection determination, we extracted genomic DNA from reference strains, quantified DNA concentration with NanoDrop 2000C (Thermo Fisher Scientific, Waltham, MA, USA), calculated copy-number/volume using dermatophyte genome sizes, and diluted DNA samples to as far as a single genome in RPA reactions.

The TwistAmp Basic Kit (TwistDX, Cambridge, UK) was used for RPA amplification. The target DNA was prepared as per the above scheme, and sterile water was used as a negative template control (NTC). The final reaction system for the quintuple RPA-EuNP-LFSBs experiment was 50 μL containing 25 μL of 2× reaction buffer, 2 μL of each of the forward primers and reverse primers (10 μM) for the five target pathogenic bacteria and 0.5 μL of each of the templates. The mixture was added to the lyophilized enzyme precipitate and mixed well. Then, 2.5 μL of 14 mM Mg²⁺ was added to the cap of the tube. The RPA reaction was incubated at 37 °C for 25 min. After the reaction was completed, the amplification products were promptly transferred into ice to stop the reaction.

All primers were ordered in Sangon Biotech (Shanghai, China), and the detailed sequences were listed in Supplementary Table S3. FAM-TTATT-Quencher used in fluorescent reporter assay was synthesized by Integrated DNA Technologies (IDT); FAM-TTATT-Biotin used in lateral flow strip test and probe in qPCR assay were ordered in TaKaRa Bio Inc. (Dalian, China). T7 RNA polymerase and NEBuffer 2.1 were purchased from New England Biolabs (MA, USA). The TwistAmp® Basic kit and Milenia HybriDetect 1 were purchased from TwistDx (Cambridge, UK). 2× AceQ qPCR Probe Master Mix and Phanta High-fidelity DNA polymerase were purchased from Vazyme (Nanjing, China).