Establishment and application evaluation of a rapid visualization detection method for Schistosoma japonicum specific nucleic acid fragments based on RPA-CRISPR/Cas12a technology

doi:10.12140/j.issn.1000-7423.2024.05.007

Abstract

Abstract:

Objective To develop a rapid visualization method for specific nucleic acid fragments of Schistosoma japonicum using the recombinase polymerase amplification (RPA) technology combined with clustered regularly interspaced short palindromic repeats-associated 12a protein (CRISPR/Cas12a), and to preliminarily evaluate its application value. Methods RPA primers, single-stranded DNA (ssDNA) reporter and CRISPR RNA (crRNA) were designed to target the SjCHGCS20 sequence (GenBank: FN356222.1). The concentrations of T4 recombinase Y, T4 recombinase X, T4 single-stranded binding protein and creatine kinase were optimized in the RPA reaction system, along with the RPA reaction temperature and time. A one-tube RPA-CRISPR/Cas12a method was established by adding the optimized RPA amplification assays, mineral oil and Cas12a reaction system to a reaction tube. After RPA amplification under optimized conditions, the Cas12a reaction was initiated by mixing with the RPA amplification products. The bright green fluorescence signal, indicative of a positive result, was visualized following the CRISPR/Cas12a-mediated cleavage reaction. The sensitivity of the RPA-CRISPR/Cas12a method was evaluated by detecting various amounts of S. japonicum genomic DNA (10⁰, 10^-1, 10^-2, 10^-3, 10^-4, 10^-5 and 10^-6 ng). The assay specificity was assessed by detecting genomic DNA (1 ng total) from S. japonicum, S. mansoni, Paragonimus westermani and Clonorchis sinensis. The effectiveness of the method was further evaluated using mice dummy positive sera containing S. japonicum genomic DNA at final concentrations of 0.2, 2, 20 and 200 ng/ml, as well as sera from mice infected with S. japonicum on day 3, 7, 21, 28, 35 and 42 post-infection. Results The optimized RPA reaction system utilized final concentrations of 50 ng/μl for T4 recombinase Y, 440 ng/μl for T4 recombinase X, 200 ng/μl for T4 single-stranded binding protein and 120 ng/μl for creatine kinase, and the optimized reaction condition was 40 ℃ for 30 min. Sensitivity evaluation of established RPA-CRISPR/Cas12a method indicated the reaction products were positive when the S. japonicum genomic DNA content exceeded 10^-5 ng. Specificity evaluation indicated the reaction product of S. japonicum genomic DNA was positive, and the reaction products of S. mansoni, P. westermani and C. sinensis genomic DNA were negative. The results of mice dummy positive sera showed that the reaction products of S. japonicum genomic DNA concentrations of 2, 20 and 200 ng/μl were positive, and the reaction product of 0.2 ng/ml was weakly positive. The reaction products of sera from mice infected with S. japonicum on day 28 and 42 post-infection were positive, and the reaction product of sera on day 35 was weakly positive. Conclusion The study successfully established a one-tube RPA-CRISPR/Cas12a method for detecting the SjCHGCS20 sequence. The method has a lowest detectable limit of 10^-5 ng for S. japonicum genomic DNA, with no cross-reaction with genomic DNA from S. mansoni, P. westermani or C. sinensis. It is rapid, sensitive, specific and simple to operate, allowing for visual observation of results under blue light, and holds potential for field diagnosis of schistosomiasis japonica.

Key words: Schistosoma japonicum, Recombinase polymerase amplification, CRISPR, Visualization detection

CLC Number:

R383.24

XU Guolei, FENG Yanye, HU Wei. Establishment and application evaluation of a rapid visualization detection method for Schistosoma japonicum specific nucleic acid fragments based on RPA-CRISPR/Cas12a technology[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2024, 42(5): 608-614.

Figures/Tables 5

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