Establishment of recombinase polymerase amplification technique for rapid detection of Schistosoma japonicum nucleic acid

doi:10.12140/j.issn.1000-7423.2020.03.006

Abstract

Abstract:

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.

Key words: Schistosoma japonicum, Recombinase polymerase amplification, Isothermal amplification, Oncomelania hupensis

CLC Number:

R383.24

WANG Sheng-lin, DENG Wang-ping, LI Yin-long, WANG Li-ping, ZHANG Li-juan, LV Shan, XU Jing. 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.

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References 30

[1]	Colley DG, Bustinduy AL, Secor WE, et al. Human schistosomiasis[J]. Lancet, 2014,383(9936):2253-2264. doi: 10.1016/S0140-6736(13)61949-2 pmid: 24698483
[2]	Zhang LJ, Mwanakasale V, Xu J, et al. Diagnostic performance of two specific Schistosoma japonicum immunological tests for screening Schistosoma haematobium in school children in Zambia[J]. Acta Trop, 2020,202:105285. doi: 10.1016/j.actatropica.2019.105285 pmid: 31786108
[3]	Dang H, Jin JN, Xu J, et al. Surveillance of schistosomiasis in People’s Republic of China in 2015[J]. Chin J Schisto Control, 2017,29(3):273-280. (in Chinese)
	( 党辉, 金嘉宁, 许静, 等. 2015年全国血吸虫病监测分析[J]. 中国血吸虫病防治杂志, 2017,29(3):273-280.)
[4]	Qin ZQ, Xu J, Feng T, et al. Field evaluation of a loop-mediated isothermal amplification (LAMP) platform for the detection of Schistosoma japonicum infection in Oncomelania hupensis snails[J]. Trop Med Infect Dis, 2018,3(4):124-133.
[5]	Yan J, Hu T, Lei ZL. The endemic situation and challenges of major parasitic diseases in China[J]. Chin J Parasitol Parasit Dis, 2015,33(6):412-417. (in Chinese)
	( 严俊, 胡桃, 雷正龙. 全国重点寄生虫病的防控形势与挑战[J]. 中国寄生虫学与寄生虫病杂志, 2015,33(6):412-417.)
[6]	Yang K, Li SZ. Application of big data mining technology in monitoring and early-warning of schistosomiasis[J]. Chin J Parasitol Parasit Dis, 2015,33(6):461-465. (in Chinese)
	( 杨坤, 李石柱. 大数据挖掘技术应用于血吸虫病监测预警研究的探讨[J]. 中国寄生虫学与寄生虫病杂志, 2015,33(6):461-465.)
[7]	Wang W, Li YZ, Li HJ, et al. Immunodiagnostic efficacy of detection of Schistosoma japonicum human infections in China: a meta analysis[J]. Asian Pac J Trop Med, 2012,5(1):15-23. doi: 10.1016/S1995-7645(11)60238-1 pmid: 22182637
[8]	Wang SP, He X, Zhou YF. Demand for and the development of detection techniques for source of schistosome infection in China[J]. Chin J Parasitol Parasit Dis, 2015,33(6):456-460. (in Chinese)
	( 汪世平, 何鑫, 周云飞. 我国血吸虫病传染源快速检测技术的需求与发展[J]. 中国寄生虫学与寄生虫病杂志, 2015,33(6):456-460.)
[9]	Zhao S, Liu YH, Li T, et al. Rapid detection of Schistosoma japonicum specific gene fragment by recombinase aided isothermal amplification combined with fluorescent probe[J]. Chin J Parasitol Parasit Dis, 2019,37(1):23-27. (in Chinese)
	( 赵松, 刘燕红, 李婷, 等. 结合重组酶介导的核酸等温扩增和荧光探针快速检测日本血吸虫基因片段[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(1):23-27.)
[10]	Dang-Trinh MA, Angeles JMM, Moendeg KJ, et al. Utilization of real time PCR for the assessment of egg burden in the organs of Schistosoma japonicum experimentally infected mice[J]. Exp Parasitol, 2018,189:61-65.
[11]	Zhang X, He CC, Liu JM, et al. Nested-PCR assay for detection of Schistosoma japonicum infection in domestic animals[J]. Infect Dis Poverty, 2017,6(1):86-92.
[12]	Zheng WB, Wu YD, Ma JG, et al. Recombinase polymerase amplification and its applications in parasite detection[J]. Chin J Parasitol Parasit Dis, 2015,33(5):382-386. (in Chinese)
	( 郑文斌, 吴耀东, 马剑钢, 等. 重组酶聚合酶扩增技术及其在寄生虫检测中的应用[J]. 中国寄生虫学与寄生虫病杂志, 2015,33(5):382-386.)
[13]	Lai MY, Ooi CH, Lau YL. Rapid detection of Plasmodium knowlesi by isothermal recombinase polymerase amplification assay[J]. Am J Trop Med Hyg, 2017,97(5):1597-1599. pmid: 28820700
[14]	Daher RK, Stewart G, Boissinot M, et al. Recombinase polymerase amplification for diagnostic applications[J]. Clin Chem, 2016,62(7):947-958. doi: 10.1373/clinchem.2015.245829 pmid: 27160000
[15]	Liu W, Liu HX, Zhang L, et al. A novel isothermal assay of Borrelia burgdorferi by recombinase polymerase amplification with lateral flow detection[J]. Int J Mol Sci, 2016,17(8):1250.
[16]	Feng T, Qin ZQ, Xu J, et al. Efficacy evaluation of a loop mediated isothermal amplification technique in detection of DNA of Schistosoma japonicum eggs in fecal samples[J]. Chin J Parasitol Parasit Dis, 2017,35(3):230-234. (in Chinese)
	( 冯婷, 秦志强, 许静, 等. 环介导等温扩增法检测粪样中日本血吸虫虫卵DNA的效果评估[J]. 中国寄生虫学与寄生虫病杂志, 2017,35(3):230-234.)
[17]	Zhu HQ, Xu J, Zhu R, et al. Comparison of the miracidium hatching test and modified Kato-Katz method for detecting Schistosoma japonicum in low prevalence areas of China[J]. Southeast Asian J Trop Med Public Health, 2014,45(1):20-25.
[18]	Yue Y, Zhang JZ, Zhang MJ. Application of NASBA and RPA in detection of pathogenic bacteria[J]. Chin J Epidemiol, 2019,40(8):1018-1022. (in Chinese)
	( 岳苑, 张建中, 张茂俊. NASBA和RPA两种等温扩增技术在病原茵检测中的应用研究[J]. 中华流行病学杂志, 2019,40(8):1018-1022.)
[19]	Piepenburg O, Williams CH, Stemple DL, et al. DNA Detection using recombination proteins[J]. PLoS Biol, 2006,4(7):e204. pmid: 16756388
[20]	Zhang HC, Wei YY, Yang ZF, et al. Establishment of RPA isothermal detection method for African classical swine fever virus[J]. Mod J Animal Husb Vet Med, 2019(11):13-16. (in Chinese)
	( 张皓淳, 魏园园, 杨作丰, 等. 非洲猪瘟病毒RPA等温检测方法的建立[J]. 现代畜牧兽医, 2019(11):13-16.)
[21]	Kersting S, Rausch V, Bier FF, et al. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis[J]. Malar J, 2014,13:99. pmid: 24629133
[22]	Rosser A, Rollinson D, Forrest M, et al. Isothermal recombinase polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection[J]. Parasit Vectors, 2015,8:446. pmid: 26338510
[23]	Poulton K, Webster B. Development of a lateral flow recombinase polymerase assay for the diagnosis of Schistosoma mansoni infections[J]. Anal Biochem, 2018,546:65-71.
[24]	Wang SL, Wang LP, Wu LL, et al. Diagnostic value of nucleic acid detection in schistosomiasis japonica: a meta-analysis[J]. Chin J Schisto Control, 2020,32(1):15-22. (in Chinese)
	( 王盛琳, 王丽萍, 吴铃铃, 等. 核酸检测技术对日本血吸虫病诊断价值的meta分析[J]. 中国血吸虫病防治杂志, 2020,32(1):15-22.)
[25]	Crannell ZA, Castellanos-Gonzalez A, Irani A, et al. Nucleic acid test to diagnose cryptosporidiosis: lab assessment in animal and patient specimens[J]. Anal Chem, 2014,86(5):2565-2571. pmid: 24479858
[26]	Kumagai T, Lu SH, Wang TP, et al. Detection of early and single infections of Schistosoma japonicum in the intermediate host snail, Oncomelania hupensis, by PCR and loop-mediated isothermal amplification (LAMP) assay[J]. Am J Trop Med Hyg, 2010,83(3):542-548. doi: 10.4269/ajtmh.2010.10-0016 pmid: 20810818
[27]	Sun K, Xing WW, Yu XL, et al. Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum[J]. Parasit Vectors, 2016,9:476.
[28]	Xing WW, Yu XL, Feng JT, et al. Field evaluation of a recombinase polymerase amplification assay for the diagnosis of Schistosoma japonicum infection in Hunan Province of China[J]. BMC Infect Dis, 2017,17:164.
[29]	Zhao S, Li T, Yang K, et al. Establishment of a recombinase-aided isothermal amplification technique to detect Schistosoma japonicum specific gene fragments[J]. Chin J Schisto Control, 2018,30(3):273-277, 306. (in Chinese)
	( 赵松, 李婷, 杨坤, 等. 重组酶介导的日本血吸虫特异性基因片段核酸等温扩增检测方法的建立[J]. 中国血吸虫病防治杂志, 2018,30(3):273-277, 306.)
[30]	Cheng G, Li D, Zhuang DF, et al. The influence of natural factors on the spatio-temporal distribution of Oncomelania hupensis[J]. Acta Trop, 2016,164:194-207.