CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >
Development and preliminary evaluation of a rapid visualization detection method for circulating nucleic acids of Schistosoma japonicum based on RPA-LFD
Received date: 2020-03-13
Online published: 2020-07-07
Supported by
National Science and Technology Major Project(2018ZX10101002-002);National Science and Technology Major Project(2018ZX10004222-004);the 60th Batch of General Financial Grant from the China Postdoctoral Science Foundation(2016M600111)
Objective To develop a rapid visualization method for specific nucleic acid fragments of Schistosoma japonicum by combining recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) methods, and evaluate its application value in detecting circulating nucleic acid of Schistosoma spp. in infected mouse serum. Methods RPA primers and probe were designed to target the S. japonicum non-long terminal repeat retrotransposons SjCHGCS19. RPA was performed using S. japonicum genomic DNA as the template, and the amplification product was examined by the LFD method. After optimizing the RPA reaction temperature and time, the RPA-LFD assay was established. The sensitivity of RPA-LFD assay was evaluated by detecting 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7 ng of S. japonicum genomic DNA; and the assay specificity was evaluated by detecting genomic DNA from S. japonicum, S. haematobium, S. mansoni, Paragonimus westermani and Clonorchis sinensis. Mouse dummy positive sera containing 0.01, 0.1, 1, 10 or 100 ng of S. japonicum adult worm genomic DNA were prepared, and mouse experiment was conducted by infection with 40 S. japonicum cerariae, from which tail vein serum was obtained before infection and on days 7, 21 and 35 after infection. Then, circulating DNA was extracted from mice dummy positive sera and infected mice sera, and examined by the RPA-LFD method, to evaluate the method’s feasibility and value of early detection in assaying specific schistosome nucleic acids in serum samples. Results The RPA-LFD assay was established to rapidly detect SjCHGCS19 repeat sequence with visualized results. The target fragments could be detected in 10 min, at 30-45 ℃. The optimal reaction condition was 39 ℃ for 20 min. The lowest detectable limit for S. japonicum adult worm genomic DNA was 10-6 ng (1 fg). Evaluation of the specificity indicated that the RPA-LFD method showed cross-reaction with the genomic DNA of S. mansoni and S. haematobium, but not with that of P. westermani and C. sinensis. The RPA-LFD assay was able to detect 0.01-100 ng of schistosome DNA fragment in mice dummy positive sera, and the free SjCHGCS19 DNA fragment in the infected mice sera on days 7, 21 and 35 after infection. Conclusion A RPA-LFD method was established for rapid visualizing detection of circulating nucleic acids of S. japonicum. This method is highly sensitive and has potential applicability for early detection of schistosome infection.
Wang-ping DENG , Qing-hua HONG , Bin XU , Sheng-lin WANG , Li-ping WANG , Jing XU , Wei HU , Xiao-nong ZHOU . Development and preliminary evaluation of a rapid visualization detection method for circulating nucleic acids of Schistosoma japonicum based on RPA-LFD[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020 , 38(3) : 286 -292 . DOI: 10.12140/j.issn.1000-7423.2020.03.005
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