CRISPR/Cas12a-based on-site diagnostics of Cryptosporidium parvum IId-subtype-family from human and cattle fecal samples

Abstract

Background: Cryptosporidium parvum is an enteric protozoan parasite with zoonotic importance and can cause cryptosporidiosis in humans as well as domestic and wild animals worldwide. The IId subtype family (SF) is one of the most prevalent subtypes of C. parvum. Some clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been developed to detect nucleic acid with high flexibility, sensitivity and specificity.

Methods: By integrating recombinase polymerase amplification and the Cas12a/crRNA trans-cleavage system (termed ReCTC), we established end-point diagnostics by observing fluorescence readouts with the naked eye under blue light and on-site diagnostics using a lateral flow strip (LFS) biosensor.

Results: Our ReCTC-based diagnoses can detect as little as a single copy of a cloned C. parvum 60-kDa glycoprotein (GP60) gene, 10 oocysts per gram (OPG), clinical fecal sample without tedious extraction of genomic DNA and have no cross-reactivity with other SFs of C. parvum or other common enteric parasitic protozoa.

Conclusions: This study provided a new strategy for direct identification of the IId SF of C. parvum free of highly trained operators and expensive special equipment.

Keywords: CRISPR/Cas12a; Cryptosporidium parvum; On-site detection; Recombinase polymerase amplification; Visualized detection.

Fig. 1

Absorbance curves of purified crRNA. The crRNA was transcribed from crDNA annealed from two reverse complementary single-strand oligonucleotides. The transcribed crRNA dealt with DNase I and was purified using the NucAway™ Spin Column

Fig. 2

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 diagnosis 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

Fig. 3

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 Real-time fluorescence intensity curves of the ReCTC-based detection involving FAM-TTATT-BHQ1 reporter

Fig. 4

Optimization of the 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 wwew eliminated with 20 nM FAM-TTATT-biotin ssDNA reporter or higher concentrations

Fig. 5

Sensitivity of the ReCTC-based detection. 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. 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 LFS assay (b, d) were indicated on the LFS pads

Fig. 6

Specificity of the ReCTC-based detection. Recombinant pUC57 plasmid DNA containing 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 plasmids DNA was added

Fig. 7

Validation of ReCTC-based detection of C. parvum IId SF in clinical cattle samples. Clinical fecal samples from 30 dairy cattle were tested by a conventional nested PCR sequencing method, b our ReCTC-based fluorescence and c LFS detection. Both the ReCTC-based fluorescence and LFS detection agreed 100% with the conventional nested PCR sequencing method

Fig. 8

Validation of ReCTC-based detection of C. parvum IId SF in 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 a ReCTC-based fluorescence and b LFS detection. N negative control