关键词: 日本血吸虫, 环介导等温扩增, CRISPR/Cas12a

Abstract:

Objective To develop a highly sensitive and specific assay for rapid detection of Schistosoma japonicum nucleic acid fragments by means of loop-mediated isothermal amplification (LAMP) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) system. Methods The Sj28S ribosomal gene fragment was selected as the target sequence, and LAMP primers, single stranded DNA (ssDNA), and CRISPR RNA (crRNA) were designed. The concentrations of each component was determined, and the optimal reaction system and conditions were screened to establish the LAMP-CRISPR/Cas12a assay for rapid fluorescent detection of S. japonicum nucleic acids. The sensitivity was evaluated using different concentrations of S. japonicum genomic DNA (1 ng/μl,100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, 10 fg/μl, 1 fg/μl) and recombinant plasmids containing the targeted Sj28S ribosomal gene fragment (10⁶, 10⁵, 10⁴, 10³, 10², 10¹, 10⁰, 10^-1 copies/μl), and the specificity was assessed using genomic DNA from S. japonicum, S. mansoni, Clonorchis sinensis, Fasciola gigantica, Paragonimus westermani, Angiostrongylus cantonensis, Echinococcus multilocularis, Neotricula aperta infected with S. mekongi, and Oncomelania hupensis with and without S. japonicum infections. Mice were infected with 40, 20 and 10 cercariae of S. japonicum, and mouse fecal samples were collected 3 to 6 weeks post-infection and plasma samples were collected 3 days and 1 to 6 weeks post-infection. The efficiency of the LAMP-CRISPR/Cas12a assay was examined for detection of stool and plasma DNA samples from mice infected with S. japonicum with qPCR assay as a parallel control. Results The LAMP-CRISPR/Cas12a assay was established based on crRNA-2, and the optimal final concentrations of ssDNA, crRNA, and Cas12a were 400, 200, and 200 nmol/L, respectively, with pre-amplification duration of 30 minutes. The lowest detection limit of this assay was 10 fg/μl for the genome of adult S. japonicum, and 1 copy/μl for the recombinant plasmids, and the assay had no cross reaction with genomic DNA from S. mansoni, C. sinensis, F. gigantica, P. westermani, A. cantonensis, E. multilocularis, N. aperta infected with S. mekongi, and O. hupensis without S. japonicum infections. The positive rates of LAMP-CRISPR/Cas12a and qPCR assays were 85.42% (41/48) and 51.02% (175/343) for detection of the pooled fecal samples from S. japonicum-infected mice, respectively (χ² = 8.71, P < 0.05), and the LAMP-CRISPR/Cas12a assay detected fecal samples from S. japonicum-infected mice as early as 3 weeks post-infection, which was earlier than qPCR assay that detected fecal samples from infected mice 4 weeks post-infection. The positive rates of LAMP-CRISPR/Cas12a and qPCR assays were 58.3% (28/48) and 27.11% (93/343) for detection of plasma samples from S. japonicum-infected mice, respectively (χ² = 9.77, P < 0.05), and both LAMP-CRISPR/Cas12a and qPCR assays detected circulating Schistosoma DNA in the plasma of S. japonicum-infected mice as early as 3 days post-infection, with positive rates of 53.06% (26/49) and 22.45% (11/49), respectively (χ² = 9.77, P < 0.05). Conclusion A simple, rapid and highly sensitive, and specific LAMP-CRISPR/Cas12a assay has been developed for rapid, early detection of S. japonicum nucleic acids, which is a promising new tool for field diagnosis of schistosomiasis.

Key words: Schistosoma japonicum, Loop-mediated isothermal amplification, CRISPR/Cas12a