Additional file 1 of CRISPR/Cas12a-based on-site diagnostics of Cryptosporidium parvum IId-subtype-family from human and cattle fecal samples
Creators: name: Yu, Fuchang
name: Zhang, Kaihui
name: Wang, Yilin
name: Li, Dongfang
name: Cui, Zhaohui
name: Huang, Jianying
name: Zhang, Sumei
name: Li, Xiaoying
name: Zhang, Longxian
Publisher: name: figshare
Year: 2021
Subjects: subject: Space Science
subject: Physical Sciences not elsewhere classified
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Environmental Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Figure S1. Absorbance curves of purified crRNA. The crRNA was transcribed from crDNA annealed from two reverse complementary single-strand oligonucleotides. The transcribed crRNA was treated with DNase I and was purified using the NucAway™ Spin Column. Figure S2. Schematic of the RPA and CRISPR-Cas12a-based detection assay. A. Diagram of Cryptosporidium parvum chromosome 6 showing primers, target sequence and crRNA. RPA primers are indicated by black rectangles; the PAM and target sequences are represented by red and blue rectangles, respectively. B. Schematic of ReCTC-based diagnostic workflow. The RPA amplicon is used directly as the input of the ReCTC-based detection, and a ternary complex forms if the target DNA exists. F, fluorophore; Q, quencher; B, biotin; F, FAM. Figure S3. Feasibility verification of the ReCTC-based detection. A. ReCTC-based fluorescence reaction products showed no signal under visible light. B. Obvious fluorescence signal can be observed under UV light by the naked eye. P1 and P2: positive results; N1 and N2: negative results. C. The real-time fluorescence intensity curves of the ReCTC-based detection involving FAM-TTATT-BHQ1 reporter. Figure S4. Optimization of reporter concentration for the ReCTC-based LFS detection. Various concentrations (200, 100, 50, 20, 15, 10, 5 nM) of FAM-TTATT-biotin ssDNA reporter were tested to avoid false-positive and -negative results. The concentrations used were labeled on the LFS pads, and false-positive results were eliminated with 20 nM or higer FAM-TTATT-biotin ssDNA reporter concentrations. Figure S5. Sensitivity of the ReCTC-based detection. A, B. Sensitivity test of ReCTC-based fluorescence (A) and LFS (B) assay using cloned recombinant plasmid DNA. The LOD of both the fluorescence and LFS assay was determined as 1.0 × 10-18 M cloned recombinant plasmid DNA. A1–A8: The concentrations of cloned recombinant plasmid DNA were 1.0 × 10-12, 1.0 × 10-15, 1.0 × 10-18, 1.0 × 10-19, 1.0 × 10-20, 1.0 × 10-21, 1.0 × 10-22, 1.0 × 10-23 M, respectively. C, D. Sensitivity test of ReCTC-based fluorescence (C) and LFS (D) assay using crude DNA extracted from purified oocysts. The LOD of both the fluorescence and LFS assay was determined as one and ten oocysts per milliliter, respectively. C1–C8: The numbers of oocysts per milliliter were equivalent to 1 × 105, 1 × 104, 1 × 103, 1 × 102, 1 × 101, 1, 0.1 and 0, respectively. The concentrations of cloned recombinant plasmid DNA and the numbers of oocysts per milliliter used in the sensitivity test of the LFS assay (B, D) were indicated on the LFS pads. Figure S6. Specificity of the ReCTC-based detection. Recombinant pUC57 plasmid DNA containing the gp60 gene of IIa, IIb, IIc, IId, IIe and IIf SFs of C. parvum (1–6) and genomic DNA of C. andersoni, C. hominis, C. meleagridis, C. muris, C. bovis, C. ryanae, Enterocytozoon bieneusi, Giardia duodenalis, Blastocystis hominis and Cyclospora cayetan (7–16) were included. A. Specificity test of the ReCTC-based fluorescence detection assay. Only the sample of C. parvum IId SF exhibited a strong fluorescence signal. B. Specificity test of the ReCTC-based LFS detection assay. A clear test line was observed only on the LFS where C. parvum IId SF recombinant pUC57 plasmid DNA was added. Figure S7. ReCTC-based detection of C. parvum IId SF on clinical cattle samples. Clinical fecal samples from 30 dairy cattle were tested by a conventional nested PCR sequencing method (A) and our ReCTC-based fluorescence (B) and LFS (C) detection. Both the ReCTC-based fluorescence and LFS detection agreed 100% with the conventional nested PCR sequencing method. Figure S8. ReCTC-based detection of C. parvum IId SF on positive clinical human samples. Clinical human fecal DNA samples collected from inpatients that had been identified as positive for C. parvum IIdA19G1 were subjected to ReCTC-based fluorescence (A) and LFS (B) detection. N: negative control. Table S1. Nucleotide sequences used in this study. Table S2. Partial sequences of C. parvum gp60 gene of six subtype families (IIa–IIf) cloned into the pUC57 vectors.