CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood

Abstract

Background: Toxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity.

Methods: This study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529 bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection.

Results: The results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/μl for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5 days post infection (dpi).

Conclusions: This study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field.

Keywords: CRISPR/Cas12a; LFS; Mouse blood; Nucleic acid testing; T. gondii.

Fig. 1

Establishment of CRISPR/Cas12a combined with RPA for detection of Toxoplasma gondii. a DETECTR workflow of T. gondii. b Comparison of different crRNAs targeting B1and 529 RE by fluorescence assay. c Screening of RPA primers for B1-L3. d Screening of RPA primers for 529 RE-L3. e Optimization of the concentration of Cas12a and crRNA

Fig. 2

Sensitivity of the DETECTR system: (a) B1-DETECTR and (b) 529 RE-DETECTR reaction with plasmid standards. The tube shows the fluorescence signal of the sample under UV light. 1–8 represent 3000 cp/μl, 300 cp/μl, 30 cp/μl, 3 cp/μl, 1.5 cp/μl, 0.3 cp/μl, 0.03 cp/μl, negative control. c B1-DETECTR and d529 RE-DETECTR reaction with T. gondii tachyzoite DNA. e B1-nested PCR and f 529 RE-nested PCR reaction with T. gondii tachyzoite DNA. 1–7 represent 10,000, 1000, 100, 10, 1, 0.1, 0.01 tachyzoite DNA per reaction. ****P < 0.0001; ns, not significant

Fig. 3

Specificity of the DETECTR system: (a) B1-DETECTR and (b)529 RE-DETECTR reaction of DNA of four different strains of T. gondii; all four strains can be detected. NC, negative control. c B1-DETECTR and d529 RE-DETECTR reaction of four types of parasite DNA, except DNA of T. gondii, could not be detected. NC, negative control; ***P < 0.001; ****P < 0.0001; ns, not significant

Fig. 4

Analysis results of blood samples from Toxoplasma gondii-infected mice. a B1-DETECTR and c 529 RE-DETECTR reaction with mouse blood DNA. 1 dpi, 1 day post-infection mice #1; NS-1, normal saline #1; PC, positive control; NC, negative control; b B1-nested PCR and d 529 RE-nested PCR reaction with mouse blood DNA. 1–15 represent sample mouse blood samples collected 1, 3, 5 days post injection; 16–20 represent samples after saline injection; 21 represents positive control; 22 represents negative control

Fig. 5

DETECTR-based LFS detection of T. gondii. a Principle of LFS. b B1-LFS and c 529 RE-LFS reaction with Specificity detection. 1–5 represent Toxoplasma gondii, Cryptosporidium parvum, Babesia, Plasmodium, negative control. d B1-LFS and e 529 RE-LFS reaction with mouse blood DNA. 1–5 represent 5 days post injection; 6–8 represent samples after saline injection; 9 represents positive control, 10 represents negative control