Abstract:

Objective To develop a method for rapid detection of Clonorchis sinensis metacercaria based on the polymerase chain reaction (PCR) technology combined with clustered regularly interspaced short palindromic repeats-associated 12a protein (CRISPR/Cas12a). Methods PCR amplification was performed using internal transcribed spacer (ITS) primers with the C. sinensis genomic DNA as template. The CRISPR reaction system was prepared by mixing PCR amplification product, Cas12a protein, CRISPR-derived RNA (crRNA), and single stranded DNA (ssDNA) reporter molecule, subsequently, the mixture was observed under ultraviolet light to verify the feasibility of the detection system, in which the ssDNA reporting molecule concentration was optimized by designing 5 gradients (100, 200, 300, 400, 500 nmol/L), and the Cas12a/crRNA ratio was optimized by formulating 5 proportional gradients (1.0 : 1, 1.5 : 1, 2.0 : 1, 2.5 : 1, 3.0 : 1) while maintaining crRNA concentration at 50 nmol/L, while the fluorescence signals were detected by using a fluorescence quantitative PCR instrument. The sensitivity of the detection system was evaluated through PCR and CRISPR reaction using the recombinant plasmid diluted to 10 different concentrations (10^-2-10⁷ copies/μl) to examine the reacted fluorescence signal. In addition, the genomic DNA extracted from C. sinensis metacercaria, Metorchis orientalis metacercaria, Spirometra erinaceieuropaei plerocercoid, Anisakis pegreffii and Taenia asiatica used as the template to assess the specificity of the detection system through PCR and CRISPR reaction, judged by checking the fluorescence signal. Samples of small-sized freshwater fish flesh from the clonorchiasis endemic area was examined by microscopy on crushed smears, from them 8 types of mixed infection were separately selected: the samples infected with mixed metacercaria of C. sinensis, M. orientalis and unknown species, metacercaria of C. sinensis and M. orientalis, C. sinensis and unknown species, M. orientalis and unknown species, C. sinensis alone, M. orientalis alone, metacercaria of unknown species alone, and none cercaria infection. These selected sample were used to extract DNA respectively for performing PCR-CRISPR/Cas12a detection, of which the reaction results were observed under ultraviolet light. The data was analyzed by using software GraphPad Prism 9.0 to perform one-way ANOVA. The comparison between groups was analyzed using graph based multiple test. Results The complete PCR-CRISPR/Cas12a detection system was able to generate fluorescence signal under ultraviolet light. When the reported molecular concentration of ssDNA is 400 nmol/L, the average fluorescence value of the reaction system is (40 786 ± 1 758) AU, which is higher than (32 029 ± 2 651) AU at the level of 300 nmol/L (P < 0.05), but no statistically significant difference (P > 0.05) was found when compared with the vlaue of (42 698 ± 4 260) AU at the level of 500 nmol/L. Therefore, the reported molecular concentration of ssDNA was 400 nmol/L. When the Cas12a/crRNA ratio was 2.0 : 1, the average fluorescence value of the reaction system was (48 950 ± 3 723) AU which was higher than (37 700 ± 3 887) AU with the ratio of 1.5 : 1 (P < 0.05) and no statistically significant difference (P > 0.05) compared to (55 630 ± 3 110) AU with the ratio of 2.5 : 1. Thus, the Cas12a/crRNA ratio was selected as 2.0 : 1. When the concentration of the recombinant plasmid was 10^-1 copies/μl, the fluorescence value of the reaction system was (39 336 ± 7 231) AU, which showed a statistically significant difference compared to the negative control (2 216 ± 743) AU (P < 0.05); when the concentration decreased to 10^-2 copies/μl, the average fluorescence value was (5 451 ± 1 957) AU, which had no statistically significant difference with that of the negative control (P > 0.05). The results of PCR showed that ITS primers could simultaneously amplify 5 kinds of genomic DNA of C. sinensis, M. orientalis, S. erinaceieuropaei, A. pegreffii, and T. asiatica. The CRISPR detection results of the same amplification products showed that only the reaction system using the genomic DNA of C. sinensis as a template generated fluorescence signals under ultraviolet light. The results of PCR-CRISPR/Cas12a detection showed that fluorescence signals were observed in four tubes containing C. sinensis metacercariae in the reaction system of 8 types of fish sample infected with the different metacercaria. Conclusion This study established a PCR-CRISPR/Cas12a method for detection of C. sinensis metacercaria, and the method is highly sensitive, specific and modularized, with significant potential for field application.

Key words: Clonorchis sinensis, Metacercaria, PCR-CRISPR/Cas12a, Molecular detection, Specificity