RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China

ABSTRACT Toxoplasma gondii, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii. The lowest limit of detection of the assay was 31 copies/μL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii, showing promise for on-site surveillance. IMPORTANCE Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii. Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.

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Reviewer #1 (Comments for the Author): In this manuscript, Sun et al developed an RPA-CRISPR/Cas12a lateral flow band assay to diagnose Toxoplasma infection.The authors applied this assay to detect Toxoplasma infection in dogs and cats from Zhejiang province, China and found that the positive rate of the infection was 4%~8%.Overall, there are several major concerns that need to be addressed, and the manuscript is poorly written with many grammatical and typographical errors.Detailed comments are listed below: 1.The RPA-CRISPR/Cas12a assay based on the Toxoplasma B1 gene has been previously developed in other studies (Lei et  al., ACS Synth.Biol., 2022; Wang et al., Parasites & Vectors, 2023).Given that the majority of the manuscript focuses on the method development, the current study seems simply to replicate the others' studies and apply the method to detect Toxoplasma infection in cats and dogs.2. The optimization of this assay is mainly performed using a plasmid containing the B1 gene, which raises concerns about the reliability and sensitivity of this method when compared to other diagnostic methods.3.In terms of the specificity of the method, the authors tested several parasite species that were phylogenetically distinct from Toxoplasma.Several coccidian parasites, such as Neospora caninum in dogs and Hammondia hammondi in cats, are commonly misdiagnosed as Toxoplasma in the clinical setting.The authors need to test if the assay can distinguish Toxoplasma from these two parasites.4. The authors need to clarify the source of stray dog and cat samples.On lines 320~ 324, the author claims these are fecal samples.However, in Figure 1 and on line 428, the author states these are blood samples.5.For all the experiments, what exactly are the negative controls?6.A lot of references in this manuscript are not appropriate and accurate.Here is the list of these references: 1, 3, 7, 8, 12, 14, 15, 19, 25, 27, 30, 31, 35, 36, 37, and 39.Please change and correct these.7. Please correct the typing errors in the manuscript, such as "high technical kills" on line 24, "eitht" on line 139 8. On line 63, please change "final host" to "definitive host".9. On line 75, time-consuming is not a major flaw of microscopic detection.Generally, microscopic detection is not sensitive and accurate.10.On line 102, the references do not have any information about Cryptosporidium and Plasmodium.
Reviewer #2 (Comments for the Author): The study provided a rapid diagnosis method using RPA-CRISPR/Cas12a-LFA for early T. gondii infection detection, which has high accuracy and sensitivity without cross reaction with other tested parasites.Therefore, the has potential for investigation the prevalence of T. gondii infection in stray dogs and cats.But I strongly suggest the authors check spelling and grammar mistakes throughout the draft, and proofread the article by natural speakers.

Reviewers' comments:
Reviewer #1: Thanks for your professional review of our article.According to your suggestions, we have made extensive corrections to our previous draft, the details of which are listed below.
In this manuscript, Sun et al developed an RPA-CRISPR/Cas12a lateral flow band assay to diagnose Toxoplasma infection.The authors applied this assay to detect Toxoplasma infection in dogs and cats from Zhejiang province, China and found that the positive rate of the infection was 4%~8%.Overall, there are several major concerns that need to be addressed, and the manuscript is poorly written with many grammatical and typographical errors.Detailed comments are listed below: 1.The RPA-CRISPR/Cas12a assay based on the Toxoplasma B1 gene has been previously developed in other studies (Lei et al., ACS Synth.Biol., 2022; Wang et al., Parasites & Vectors, 2023).Given that the majority of the manuscript focuses on the method development, the current study seems simply to replicate the others' studies and apply the method to detect Toxoplasma infection in cats and dogs.
Re: Thank you for these comments.Compared with those two papers, the main innovations of our study are as follows: Firstly, we established a visual RPA-CRISPR/Cas12a-LFA system combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye.In the studies by Lei et al. and Wang et al. study, they both used "elimination method" test strips.Li et al. [1] report that the "elimination method" test strips are less sensitive and generate false-negative results.Secondly, the developed RPA-CRISPR/Cas12a-LFA assay was used for a new epidemiological survey of the Toxoplasma gondii infection rate in stray cats and dogs in Zhejiang.
[1] Li H, Dong X, Wang Y, Yang L, Cai K, Zhang X, Kou Z, He L, Sun S, Li T, Nie Y, Li X, Sun Y.
2. The optimization of this assay is mainly performed using a plasmid containing the B1 gene, which raises concerns about the reliability and sensitivity of this method when compared to other diagnostic methods.
Re: Thank you for this comment.Plasmids have many advantages, such as convenience and stability, in the development of methods.It has been reported that it is feasible to verify the sensitivity of the detection method by constructing a B1 gene plasmid.Before starting our study, we consulted previous studies.For example, Jiang et al. [2] validated the sensitivity of the established PCR method by testing with the pMD-19T-B1 plasmid, and they applied this method to detect T. gondii in meat.
Re: Spectrum03998-23R1 (RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China) Dear Mr. Hao Sun: Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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