CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >
Establishment of recombinase polymerase amplification technique for rapid detection of Schistosoma japonicum nucleic acid
Received date: 2020-01-02
Online published: 2020-07-07
Supported by
National Science and Technology Major Project(2018ZX10101002-002);National Science and Technology Major Project(2018ZX10004222-004)
Objective To establish a sensitive, specific, simple and rapid method for detecting Schistosoma japonicum nucleic acid by using the recombinase polymerase amplification (RPA) technique. Methods Specific primers were designed to target the 28S ribosomal S. japonicum (Sj28S) gene using Primer Premier 5 software. The RPA method was established and the reaction conditions were optimized. The specificity and sensitivity of the method were tested using genomic DNA extracted from S. japonicum at different stages, S. mansoni, S. haematobium, Oncomelania hupensis infected with single-tail cercariae, Clonorchis sinensis, Fasciola gigantica, Paragonimus westermani, and Taenia saginata. The detection performance and reproducibility of the RPA method were assessed using the mixed genomic DNA from infected and non-infected O. hupensis at various ratios (1 : 10, 1 : 50, 1 : 100, 1 : 250, 1 : 500, 1 : 1 000, 1 : 2 000). Results The RPA method specifically amplified the 216-bp target gene fragment of S. japonicum. The optimal reaction conditions of RPA were at 39 ℃ for 20 min. No cross reactions occurred with the genomic DNA of S. mansoni, S. haematobium, O. hupensis infected with single-tail cercariae, C. sinensis, F. gigantica, P. westermani, T. saginata and O. hupensis with negative infection. The lowest detectable limit of the RPA method for genomic DNA of S. japonicum adults was 100 fg/μl, and for the recombinant plasmid was 100 copies/μl. In addition, the RPA method could exactly detected the target DNA of S. japonicum at different stages. Tested with mixed genomic DNA of infected and all snails at various ratios, the lowest detectable limit of the RPA method for the target DNA fragment was 1 : 1 000. The test was repeated for 5 times, and all produced consistent results, with no false negative or false positive results. Conclusion The RPA method established for detecting S. japonicum nucleic acid displays high sensitivity and specificity, and is simple and rapid to use. Thus, the method could be used for rapid detection and risk monitoring.
Sheng-lin WANG , Wang-ping DENG , Yin-long LI , Li-ping WANG , Li-juan ZHANG , Shan LV , Jing XU . Establishment of recombinase polymerase amplification technique for rapid detection of Schistosoma japonicum nucleic acid[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020 , 38(3) : 293 -298 . DOI: 10.12140/j.issn.1000-7423.2020.03.006
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