The CRISPR-Cas system has been extensively deployed to accelerate the highly efficient diagnostic toolkit with speed, specificity and sensitivity, relying on gRNA binding with target sequences on the DNA or RNA target site (Kim et al., 2021). Therefore, extensive cas proteins such as cas12 and cas13 were coupled with isothermal nucleic acid amplification for pathogen detection; for example, cas13 for SARS-CoV-2 and RAA-cas12a for African swine fever virus detection (Chandrasekaran et al., 2022a; X. Wang et al., 2020). Our findings revealed that RPA for amplification and cas12a for detecting the DNA target has been successfully harnessed to diagnose E. canis and A. platys with high specificity, sensitivity and simplicity that require minimal instrument, possibly adopting POCT.
In the present study, the suitable concentration and ratio of cas12a and gRNA to form a binary complex was a ratio of cas12a:gRNA 100:100 nM which previous studies demonstrated the difference in those factors. For instances, the optimal condition of the cas12a platform for mycoplasma contamination detection was concentration of gRNA:cas12a as 500:250 nM at 37°C for 30 min or hepatitis C virus detection as 40 nM of cas12a:gRNA with detection within 30 min. These studies applied isothermal amplification including RPA or LAMP coupled with cas12a that demonstrated 100% accuracy for detection (Kham-Kjing et al., 2022). We demonstrated the efficiency of the RPA-assisted cas12a assay for parasite detection which the method has a few processing steps including RPA for DNA amplification within 20 min, followed by detection using the cas12a assay within one hour to detect the DNA of the parasite within two hours, and requires only an incubator as the reaction can be completed at 37°C.
The RPA-assisted cas12a assay displayed explicit specificity for DNA parasites of E. canis and A. platys detection without cross-reaction to other parasites under the optimal condition, indicating that cas12a detection showed endurance with mismatch sequences or no targets from RPA amplification. The primers for the RPA process selected a similar region of 16S rRNA between E. canis and A. platys but distinguished from other hemoparasites infected in dogs or other mammals. The primer amplified E. canis and A. platys 16S rDNA, resulting in an increased amount of DNA target and then gRNA was designed from the region with species-specific sequence on the DNA target amplified. The specific gRNA of E. canis and A. platys have single nucleotide variation at the third and fifth position from the 3’ end next to PAM. The seed region of gRNA which is mismatch intolerant is at the first five nt next to PAM (Zetsche et al., 2015). Different nucleotides, especially single nucleotide polymorphism (SNP) on the seed region and the PAM site, caused both gRNAs to have specific detection based on similar amplification regions. The specificity determination showed that gRNAs can only bind with DNA targets because the fluorescence signal from the collateral activity is significantly different between DNA targets and other DNA parasites. Our previous study demonstrated that RPA combined with cas12a showed promise for species differentiation of Phyllanthus amarus from other closely relating species, based on the difference in PAM sequence and 4–5 nucleotides at the 5’ end (seed sequence) of the spacer. These features can be used to facilitate target-specific detection in the DNA region of interest of certain organisms (Buddhachat et al., 2021; Gerashchenkov et al., 2020). In addition, we evaluated the specificity of the RPA-assisted cas12a assay using either gRNA_E or gRNA_A in complex condition, with admixture of DNA templates of various hemoparasites like co-infection. Our findings exhibited that although the DNA templates were admixed with other parasites’ DNA, the assay using either gRNA_E or gRNA_A also gave the fluorescence signal in only the condition with the presence of E. canis or A. platys, respectively. This suggested that the other DNA parasites did not interfere with the assay.
However, we noted that the RPA-assisted cas12a assay using gRNA_E was incubated for longer than two hours, enabling nonspecific detection with A. platys DNA but not for the RPA-assisted cas12a using gRNA_A. Therefore, the gRNA_E showed binding DNA of A. platys in a similar region because the sequence of gRNA_E was different from the DNA of A. platys by only one base pair from the SNP of the seed region. The gRNA_E may have cross-activity with other species due to DNA sequences of different parasites being similar (Unver et al., 2003). Moreover, incubating reactions for a long-time resulted in cas-gRNA complex binding and cutting of non-specific targets that presented cleavage fluorescence signals of no targets. Consequently, the study for cas12a detection used the various optimal concentration of cas12a:gRNA and the appropriate time for detection (Buddhachat et al., 2022b; Ding et al., 2020a; Mukama et al., 2020). However, the cas12a assay can specifically detect different DNA within one hour, thereby saving time for detection. The cas12a-gRNA complex could distinguish types of parasites in one reaction; therefore, proposing a cas12a detection system for multiplex parasite detection in one-pot reaction. Previously, Cas13 was developed to detect multiplex Dengue or Zika viruses (Gootenberg et al., 2018). Future studies should investigate the collateral cleavage trans-activity properties of other cas12 proteins that can detect multiplex parasites in one-pot cas12 assay from the different color labels on the reporter.
The RPA-assisted cas12a assay using both gRNA_E and gRNA_A has a limit of detection of 100 copies of DNA target per reaction. The detection limit of the RPA-assisted cas12a assay using gRNA_E and gRNA_A compared to gel electrophoresis detection was 1,000 and 100 times greater, respectively. Furthermore, the RPA products yielded the expected amplicons and generated primer dimerization or non-specific smear bands that appeared on gel electrophoresis, leading to the interference of detection but unaffected by cas12a detection. This was unsurprising because signal amplification from the RPA reaction was initiated by the operation of the cas12a-gRNA complex for DNA target binding. Therefore, cas12a collaborated with the isothermal amplification methods extensively used for detection such as LAMP-cas12a for African swine fever and Escherichia coli detection (Lee & Oh, 2022; Yang et al., 2022). RPA-cas12a for COVID-19 and Toxoplasma gondii detection (Ding et al., 2020b; Lei et al., 2021). The sensitivity of the assay was dependent on (i) the amplicon the size of RPA (the smaller amplicon size, the more sensitive) and (ii) the condition of the cas12a assay such as the concentration of cas and gRNA used, the sequences of the reporter and the additives (Lv et al., 2021). When the result of RPA coupled with cas12a was compared with gel electrophoresis detection, the RPA-assisted cas12a assay had greater specificity and sensitivity. The limit of detection could be improved using high-efficiency ssDNA reporters to produce the fluorescence signal. A study of engineering the DNA reporter (TTATT-5C) enabled a ten-fold sensitivity increase compared with the TTATT reporter, while the optimal concentration of DNA reporter engineering clearly showed a fluorescence signal between the target and non-target in the cas12a assay (Lee et al., 2022). This study determined the background of fluorescence signals in the negative control that generated a fluorescence signal even if the DNA target disappeared. In future studies, a new ssDNA reporter should be modified to improve the performance of cas12a for trans-activity detection.
Several methods for rickettsia diagnostics provide different detection efficiencies. Microscopic observation has been used for rickettsia diagnosis with low sensitivity due to difficulty in observing blood smears when dogs become infected after two weeks (Chandrasekaran et al., 2022b). The ELISA used for rickettsia detection reported high sensitivity, with detection 14 days after inoculation, while molecular assay gave detection within seven days and the serological method gave a false positive result (Suksawat et al., 2000). Currently, the diagnosis of rickettsia includes PCR or real-time PCR. Real-time PCR for Ehrlichial species detection has limited detection at 50 and 100 copies per reaction (Cárdenas et al., 2007; Lloyd et al., 2011).
The RPA-assisted cas12a assay using gRNA_E and gRNA_A was validated with real clinical samples that showed efficiency for E. canis and A. platys detection from canine peripheral blood. The RPA enabled DNA amplification of E. canis and A. platys, and both gRNA_E and gRNA_A provided high accuracy to detect parasites owing to disappearing fluorescence signals in non-target DNA. The fluorescence signals acquired from the RPA-assisted cas12a assay can be readily observed by the naked eye under a blue LED transilluminator. The detection limit of this method was sufficient for real sample detection. Moreover, the RPA-assisted cas12a, assay results gave accuracy with the HRM assay of more than 80% for E. canis and A. platys detection. We also confirmed the results obtained from both approaches by sequencing, as a result of the sequence similarity to E. canis, with more than 90% identity. These results indicated the reliability of both methods to detect E. canis and A. platys in real blood samples. Diagnosis of the presence of parasites in canine blood diseases should be performed rapidly and accurately for precise and effective treatment of the ailments. Moreover, fast diagnosis and treatment can assist in saving the dog’s life and decrease the contagion of disease(Lauzi et al., 2016; Yuasa et al., 2017). This study articulated the optimal situation of the RPA-assisted cas12a assay to detect parasites in canine blood with specificity and sensitivity. Moreover, the method was simple and detection was fast, requiring few instruments with easy-to-interpret results. The lateral flow dipstick can be useful to interpret results for detection in the field (Xu et al., 2022; Zhang et al., 2021). However, parasite diagnostics in the field applying the RPA-assisted cas12a assay might have airborne contamination. Therefore, the hands-on process needs to be performed carefully and the reaction should be performed in a closed area. When using the RPA-assisted cas12a assay for practical POCT, efficient and rapid DNA extraction from canine blood needs to be developed such as using a dipstick, buffer or heating methods. Simple DNA extraction methods can be directly input into the RPA reaction and detected by the cas12a assay (Bereczky et al., 2005; Koontz et al., 2019; Mason & Botella, 2020).