Rapid and visual detection ofSchistosoma japonicumcirculating nucleic acids by CRISPR-Cas12a
Authors: Li Qian , Yang Shuqi , Cheng Miao , Xing Chen , Wang Xiaofeng , Zhu Yating , Tang Yunshu , Cheng Wenhui , Shen Jijia , Du Yinan 
Published: August 2023

Background

Schistosomiasis is the second most destructive parasitic disease globally. It poses a serious threat to human health if not diagnosed and treated promptly. However, the current detection methods have limitations due to their dependence on specialized equipments and personnel, and the long detection time, resulting in less-than-ideal detection outcomes. Therefore, more accurate and sensitive assays are required to aid in the control of schistosomiasis. This study aims to develop a rapid and practical method for detectingSchistosoma japonicumcirculating nucleic acid by combining the Cas12a protein in the CRISPR (Clustered regularly interspaced short palindromic repeats) system with recombinase-mediated isothermal nucleic acid amplification technology.

Methodology/Principal Findings

We created a CRISPR-Cas12a detection method for Schistosomiasis.SjR2andSjCHGCS19genes with repetitive copiesin the Schistosoma japonicumgenome were targeted for detection. The CRISPR-Cas12a detection method can achieve a detection sensitivity of 1 cp/µL, with no cross-reaction with the other three human-infecting trematode parasites. To vividly simulate clinical patients at different infection stages, we collected rabbits’ serums at 1 d, 3 d, 1 w, 3 w, 5 w, and 7 w post-infection. CirculatingSchistosoma japonicumDNA in the serum can be detected as early as 3 days post-infection. The sample and positive rates of each stage were analyzed and the CRISPR-Cas12a detection method prevailed to Nested-PCR. Additionally, our assay offers flexible results readout options, including lateral test strips.

Conclusions/Significance

This CRISPR-Cas12a detection method enables sensitive and specific detection of Schistosomiasis by detecting highly repetitiveSjR2andSjCHGCS19genes. The method was fully validated with rabbits’ serums collected at different infection stages, which can be applied to monitoring the early infection of schistosomiasis in resource-limited regions.

Author Summary

With the development of schistosomiasis prevention and control efforts in recent years, the prevalence rate and infection intensity of schistosomiasis have decreased, making early and accurate diagnoses more challenging. Combining CRISPR-Cas12a protein with isothermal amplification technology, a detection method for the multi-copy sequencesSjR2andSjCHGCS19in the genome ofSchistosoma japonicumwas developed in this study. The results demonstrated that the detection sensitivity of this method could reach 1 cp/µL, with no cross reactivity with three other fluke-like human parasites. In this study, serum samples from 15 rabbits infected withSchistosoma japonicumwere used to validate the method. The results demonstrated that rabbits could be identified as early as the third day after infection. CRISPR-Cas12a detection method has a higher positive rate than traditional Nested-PCR. In addition, lateral test strips enable direct observation of our assay results. The aforementioned research findings indicate that the CRISPR-Cas12a-based detection method can be utilized for the early diagnosis of schistosomiasis. In conjunction with the result reading method of the lateral test strips, it also provides a new method for the on-site diagnosis of schistosomiasis in environments with limited access to resources.