Primer explorer v5

All real-time RT-qPCR and RT-LAMP reactions were performed on a qPCR machine (Applied Biosystems® 7500, Thermo Fisher Scientific, UK). A single-sided MicroAmp® Optical Adhesive Film was purchased from Thermo Scientific. Polymethyl methacrylate (PMMA) plate was obtained from Wanglong Materials (Shenzhen, China). Three sets of LAMP primers were designed using LAMP primer software Primer Explorer V5 (http://primerexplorer.jp/e/intro/index.html; Eiken Chemical Co., Japan). For increasing the reliability of the results, we included the BRAC1 gene (see Table S2 for detailed sequences) as the positive control, which resulted in less possibilities of false-negative results [31 (link)]. All primers and plasmids were synthesized by Sangon Company (Shanghai, China). Extraction of RNA was based on a nucleic acid extraction kit (3DBiopharm, 20200219, China) and a nucleic acid isolation machine (3DBiopharm, NP968-C, China). All clinical samples were provided and tested at the West China Hospital, Chengdu, under the authorization of the Chinese CDC. The research on the multiplexed and point-of-care diagnosis of COVID-19 using clinical samples had been approved by the ethical committee at the West China Hospital, Chengdu, China (No. 2020 (267)).

Loop-mediated isothermal amplification primers for A. tomentosa detection were designed to target the ITS-2 sequence described earlier. Primers (Table 6) were designed using either Primer Explorer V5 (Eiken Chemical Company; https://primerexplorer.jp/e/) with default settings or CLC Genomics (Qiagen, Hilden, Germany) to manually design LAMP primers following primer design requirements as described by Nagamine and Notomi (Nagamine et al. 2002 (link); Notomi et al. 2000 ).Table 6

Loop-mediated isothermal amplification primers used for Austropeplea tomentosa detection in this study

Primer name	Primer sequence 5′-3′a	Sequence region 5′-3′
Set 1 F3	GTCTCAAGCACAAGCCGC	F3
Set 1 B3	GGGCGCCGATTTGTCAAG	B3
Set 1 FIP	GAGGAAAATTTGGCCGCCGCCCGTTGTCCGTGTTCGTC	F1,F2
Set 1 BIP	CTAACGGGCCCGCTCGTAACTCAGCGTAAGCTTCTCTCCT	B1, B2
Set 1 LF	AGAGCAAGGCGGCGT	LF
Set 1 LB	AAGCTCCAGGGTGATTGCG	LB
Set 2 F3a	TGCACGGTGTTGCCCG	F3
Set 2 B3a	GCGTAAGCTTCTCTCCTCCG	B3
Set 2 FIPa	GCGGACGTCCCGAGACGAACTGGCCCCGTGGTCTCAA	F1,F2
Set 2 BIPa	TTCCTCCTCGTCACCGCTATGCCAATCACCCTGGAGCTTGTT	B1, B2
Set 2 LF	GCGGCGGCCAAATT	LF
Set 2 LB	CGGCTCGCTCTCGCTAA	LB
Set 3 F3	GGTGGCCCCGTGGTCT	F3
Set 3 B3	CTTCTTCAATTCGTACGGGCG	B3
Set 3 FIP	AGCGGTGACGAGGAGGAAAATTCGTGTTCGTCTCGGGACGT	F1,F2
Set 3 BIP	CGTAACAAGCTCCAGGGTGATTGTTGTCAAGCGAGCGTCAGC	B1, B2
Set 3 LF	CGAGAGCAAGGCGGCGT	LF
Set 3 LB	CGGAGGAGAGAAGCTTAC	LB

^adenotes chosen primer combination used throughout study

For each gene target, we first used data from the Human Protein Atlas Project (39 (link)) to identify its most highly expressed transcriptional isoforms in our target cell type. We then used the Ensembl Genome Browser (40 (link)) to identify common regions between highly expressed isoforms, and designed LAMP primers to target these sequence regions. We designed LAMP primers using PrimerExplorer V5 software (Eiken Chemical Co., http://primerexplorer.jp/lampv5e). We found SNAPD’s sensitivity was greatly improved if we used two different sets of LAMP primers to target each transcript. In these cases, we designed primers such that little or no overlap occurred between them.

LAMP primers for SARS-CoV-2 detection were designed based on the sequences of the ORF1b region of the virus. The SARS-CoV-2 sequences available in GenBank and GISAID were aligned using BioEdit 7.0.5.3 software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) to identify conserved regions. A consensus sequence of the ORF1b region was used to design LAMP primers through Primer Explorer V5 software (Eiken; http://primerexplorer.jp/). The RT-LAMP assay requires a set of 6 primers: 2 outer primers (F3 and B3), 2 inner primers (FIP and BIP), and 2 loop primers (LF and LB). FIP consists of a complementary sequence of F1 and a sense sequence of F2, whereas BIP includes a complementary sequence of B1 and a sense sequence of B2 (10). The detailed primer sequences used for SARS-CoV-2 amplification are shown in Table 1.

Primers for RT-qLAMP were designed based on sequences retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?SeqType_s=Nucleotide, accessed on 1 September 2022). The sequences were aligned using CLC Main Workbench 21 (Qiagen). Primer sets were initially designed using Primer Explorer V5 (Eiken Chemical Co., Ltd., Tokyo, Japan; http://primerexplorer.jp/lampv5e/index.html, accessed on 1 September 2022), then analyzed using NetPrimer (Premier Biosoft, San Francisco, CA, USA; NetPrimer/AnalyzePrimerServlet">http://www.premierbiosoft.com/NetPrimer/AnalyzePrimerServlet, accessed on 1 September 2022) to verify compatibility. Primer sets included an outer forward primer (F3), an outer backward primer (B3), a forward inner primer (FIP), and a backward inner primer (BIP). To accelerate the reaction when available, loop forward (LF) and loop backward (LB) primers were designed. Detailed information regarding all primer sets is presented in Table 1. All primers were synthesized by Macrogen (Macrogen Inc., Seoul, Korea). Primer solutions for individual assays were prepared and comprised 0.2 µM F3 and B3, 1.6 µM FIP and BIP, and 0.6 µM LF and LB, then stored at −20 °C.