Total RNA was extracted from lung tissue homogenate using TRIzol reagent (Songon Biotech, Shanghai, China) according to the manufacturer’s instructions. Then, cDNA synthesis was conducted using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, K1622, Waltham, MA, United States) in a 20 μl volume of reaction mixture. Real-time PCR was performed in a total reaction volume of 10 μl, containing 5 μl PerfectStartTM Green qPCR SuperMix (TransGen Biotech, Beijing, China), 2 μl template cDNA, 1 μl of primers (Shenzhen Huada Gene Research Institute, Shenzhen, China), and 2 μl DNase/RNase-free water (Tiangen Biotech, Beijing, China). Finally, pvdQ of P. aeruginosa was chosen as the reference gene. The qRT-PCR reaction was conducted on a LightCycler® 480II Master Mix (Roche, Germany). Data from qRT-PCR experiments were analyzed using the 2®-ΔΔCT method (Yang et al., 2021 (link)). Primer sequences are listed in Table 1 .
Dnase rnase free water
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DNase/RNase-free water is a high-purity water product designed for use in molecular biology applications. It is free from detectable DNase and RNase activity, ensuring the integrity of nucleic acid samples.
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4 protocols using dnase rnase free water
1
Isolation and qRT-PCR Analysis of P. aeruginosa Gene Expression
2
SARS-CoV-2 Detection Using CRISPR-Cas13
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All oligonucleotides sequences were shown in Table S1 (Supporting information). Template oligonucleotide sequences and the plasmid of SARS-CoV, human RPP30 (Hs-RPP30), and Middle East respiratory syndrome coronavirus (MERS-CoV) were purchased from Sangon Biotech (Shanghai, China), and other oligonucleotide sequences were synthesized by ourselves. MiRcute miRNA Isolation Kit (DP501) and DNase/RNase-free water were purchased from TIANGEN Biotechnology Co. The T7 Mix (MAXI 100), Taq DNA polymerase (5000 U·mL−1), DNase I (2000 U·mL−1), 10 × DNase I buffer, 10 × NEB buffer 2.1, ProtoScript® II Reverse Transcriptase (10 000 U·mL−1), T7 RNA polymerase (50 000 U·mL−1), and RNA inhibitor (40 000 U·mL−1) were ordered from New England Biotechnology Co., Ltd (Beijing, China). The RPA kit was supplied by TwistDx (Cambridge, UK). LwCas13a (100 μM) protein was ordered from Beijing Kesin Biotechnology Co., LTD. Universal lateral flow dipstick for detection of biotin- and FITC-labeled analytes was bought from Milenia Biotec (Germany).
3
Quantification of Quorum Sensing Genes in P. aeruginosa
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Fluorescence real-time PCR was used to detect the expression of the QS genes of PAO1 in the presence of paeonol (0, 128, 256, and 512 μg/mL). Table 1 shows the primers that were used to amplify lasI, lasR, rhlI, rhlR, pqsA, pqsR, lasA, lasB, rhlA, rhlC, phzm, phzM, phzH, phzS, and pvdQ (reference gene). Total RNA was then extracted using TRIzol Reagent (Songon Biotech, Shanghai, China) according to the manufacturer’s instructions. Next, the samples were reverse-transcribed to single-stranded cDNA in a 20-μL volume of the reaction mixture using Maxima H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, United States). Then, real-time PCR was performed in a 10-μL reaction volume, containing 5 μL of PerfectStartTM Green qPCR SuperMix (TransGen Biotech, Beijing, China), 2 μL of the template cDNA, 1 μL of primers (Shenzhen Huada Gene Research Institute, Shenzhen, China), and 2 μL of DNase/RNase-free water (Tiangen Biotech, Beijing, China). The pvdQ gene was chosen as a reference gene and used to normalize the quantitative PCR data and calculate the relative differences in mRNA expression using the 2–△△CT method.
4
Multiplex CRISPR-Cas Detection via Lateral Flow
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In this study, the readout was visualized using commercially available multiplexed lateral flow strips (HybriDetect 2T, Milenia, Germany) following the manufacturer's protocol. We generated the orthogonal CRISPR-Cas12b/Cas13a detection mix as described above, except replacing the fluorescent probes with biotinylated ssDNA probe at a final concentration of 1μM and digoxin-modified ssRNA probe at a final concentration of 150 nM. Detailed probe sequences are listed in Table S3 .
After incubating the detection reaction for 15 minutes at 39°C, followed by 30 minutes at 60°C, the reaction was diluted 1:5 in DNase/RNase-Free water (TIANGEN, China). Subsequently, 10 μL of the diluted reaction mixture was directly loaded onto the sample pad area of the HybriDetect 2T lateral flow strips. The loaded strips were then placed into 80 μL of HybriDetect 2T Assay Buffer (Milenia, Germany) and incubated for 5 minutes at room temperature in an upright position. The lateral flow results were assessed by the user.
After incubating the detection reaction for 15 minutes at 39°C, followed by 30 minutes at 60°C, the reaction was diluted 1:5 in DNase/RNase-Free water (TIANGEN, China). Subsequently, 10 μL of the diluted reaction mixture was directly loaded onto the sample pad area of the HybriDetect 2T lateral flow strips. The loaded strips were then placed into 80 μL of HybriDetect 2T Assay Buffer (Milenia, Germany) and incubated for 5 minutes at room temperature in an upright position. The lateral flow results were assessed by the user.
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