CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >
Establishment and application of a multiplex recombinase-aided isothermal amplification technique for identifying Echinococcus granulosus and Echinococcus multilocularis
Received date: 2019-11-29
Online published: 2020-07-07
Supported by
National Key Research and Development Program of China(2016YFC1200500);Comprehensive Demonstration Project on Echinococciosis of Chinese Center for Disease Control and Prevention(201901)
Objective To establish a multiplex recombinase-aided isothermal amplification (mRAA) method for rapid detection of Echinococcus granulosus G1 (EgG1) and E. multilocularis (Em) DNA. Methods The mRAA rapid detection method for Echinococcus was established by designing primers based on the mitochondrial genes of EgG1 and Em as target sequences. The sensitivity of the mRAA method was assessed by amplifications of EgG1 and Em genomic DNA at concentrations of 10.00, 5.00, 1.00, 0.50, 0.10, 0.05, and 0.01 ng/μl and of pMD19-T (Simple) recombiant plasmids at 105, 104, 103, 102 and 10 copies/μl. The specificity of the mRAA method was evaluated by using genomic DNA of Taenia saginata, T. asiatica, T. multiceps, Dipylidium caninum, Toxocara canis, Trichuris trichiura Linnaeus, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Fasciola gigantica and Clonorchis sinensis as templates for amplification. To verify the reliability and feasibility, optimized mRAA method was used to examine three fecal samples of dogs with simulated infection of mixed EgG1 and Em. 19 liver tissue samples of infected animals(10 with EgG1 infection and 9 with Em infection) collected from field, 7 fecal samples of infected dogs (5 EgG1 infected and 2 Em infected) collected from field. Results The established mRAA method could specifically amplify the EgG1 and Em mitochondrial gene fragments with the sequence lengths of 250 and 500 bp, respectively. The detactable limit of EgG1 and Em genomic DNA by mRAA was 2 pg/μl, and that of the EgG1 and Em recombinant plasmids was 200 copies/μl. The mRAA method showed negative results of amplification on genomic DNA of T. saginata, T. asiatica, T. multiceps, D. caninum, T. canis, T. trichiura Linnaeus, G. lamblia, F. hepatica, P. westermani, F. gigantica and C. sinensis. It was demonstrated that the mRAA method successfully detected EgG1 and Em DNA from the fecal samples of dogs with simulated infection of mixed EgG1 and Em, the liver tissue of infected field animals, and fecal samples of infected field dogs. Conclusion The mRAA method displays evident sensitivity, specificity and reliability, and could be used for rapid detection of EgG1 and Em DNA.
Hong-rang ZHOU , Guang-yao MAO , Xiao-ling WANG , Mu-xin CHEN , Qing YU , Ying WANG , Lin Ai , Ning XIAO . Establishment and application of a multiplex recombinase-aided isothermal amplification technique for identifying Echinococcus granulosus and Echinococcus multilocularis[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020 , 38(3) : 310 -316 . DOI: 10.12140/j.issn.1000-7423.2020.03.09
| [1] | Liu H, Xiao N, Yang SJ, et al. Epidemiological characteristics of canine Echinococcus infection in Qinghai-Tibet plateau of China[J]. Chin J Schisto Control, 2017,29(2):129-138. (in Chinese) |
| [1] | ( 刘辉, 肖宁, 杨诗杰, 等. 青藏高原地区犬棘球绦虫感染的流行病学特征[J]. 中国血吸虫病防治杂志, 2017,29(2):129-138.) |
| [2] | Wu WP, Wang H, Wang Q, et al. A nationwide sampling survey on echinococcosis in China during 2012-2016[J]. Chin J Parasitol Parasit Dis, 2018,36(1):1-14. (in Chinese) |
| [2] | ( 伍卫平, 王虎, 王谦, 等. 2012-2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(1):1-14.) |
| [3] | Eckert J, Deplazes P. Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern[J]. Clin Microbiol Rev, 2004,17(1):107-135. |
| [4] | Wu WP. Prevalence and distribution of hydatidosis in China[J]. China Animal Heal, 2017,19(7):7-9. (in Chinese) |
| [4] | ( 伍卫平. 我国两型包虫病的流行与分布情况[J]. 中国动物保健, 2017,19(7):7-9.) |
| [5] | Enrico B, Peter K, Dominique AV. Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans[J]. Acta Trop, 2010,114(1):1-16. |
| [6] | Budke CM, Deplazes P, Torgerson PR. Global socioeconomic impact of cystic echinococcosis[J]. Emerging Infect Dis, 2006,12(2):296-303. |
| [7] | Torgerson PR, Keller K, Magnotta M, et al. The global burden of alveolar echinococcosis[J]. PLoS Negl Trop Dis, 2010,4(6):e722. |
| [8] | Zhang MY, Wu WP, Guan YY, et al. The analysis of disease burden of echinococcosis in China[J]. Chin J Parasitol Parasit Dis, 2018,36(1):15-19. (in Chinese) |
| [8] | ( 张梦媛, 伍卫平, 官亚宜, 等. 我国棘球蚴病疾病负担分析[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(1):15-19.) |
| [9] | Karamon J. Detection of Echinococcus multilocularis in faeces by nested PCR with the use of diluted DNA samples[J]. Pol J Vet Sci, 2014,17(1):79-83. |
| [10] | Dinkel A, Kern S, Brinker A, et al. A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species[J]. Parasitol Res, 2011,109(2):493-498. |
| [11] | Knapp J, Millon L, Mouzon L, et al. Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools[J]. Vet Parasitol, 2014,201(1/2):40-47. |
| [12] | Trachsel D, Deplazes P, Mathis A. Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA[J]. Parasitology, 2007,134(6):911-920. |
| [13] | Liu CN, Lou ZZ, Li L, et al. Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus using a multiplex PCR assay[J]. PLoS Negl Trop Dis, 2015,9(9):e0004084. |
| [14] | Fan XX, Zhao YG, Li L, et al. Research progress of recombinant enzyme polymerase amplification (RPA) in rapid detection of diseases[J]. China Animal Heal Insp, 2016,33(8):72-77. (in Chinese) |
| [14] | ( 樊晓旭, 赵永刚, 李林, 等. 重组酶聚合酶扩增技术在疾病快速检测中的研究进展[J]. 中国动物检疫, 2016,33(8):72-77.) |
| [15] | Santiago-Felipe S, Tortajada-Genaro LA, Puchades R, et al. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis[J]. Anal Chim Acta, 2014,811:81-87. |
| [16] | Piepenburg O, Williams CH, Stemple DL, et al. DNA detection using recombination proteins[J]. PLoS Biol, 2006,4(7):e204. |
| [17] | Abd El Wahed A, El-Deeb A, El-Tholoth M, et al. A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus[J]. PLoS One, 2013,8(8):e71642. |
| [18] | LV B, Cheng HR, Yan QF, et al. Recombinase-aid amplification: a novel technology of in vitro rapid nucleic acid amplification[J]. Sci Sin Vitae, 2010,40(10):983-988. (in Chinese) |
| [18] | ( 吕蓓, 程海荣, 严庆丰, 等. 用重组酶介导扩增技术快速扩增核酸[J]. 中国科学: 生命科学, 2010,40(10):983-988.) |
| [19] | Li J, Macdonald J. Advances in isothermal amplification: novel strategies inspired by biological processes[J]. Biosens Bioelectron, 2015,64:196-211. |
| [20] | Meng QH. Progress in the diagnosis and treatment of hydatidosis in China[J]. Chin J Urban Rural Enterp Hyg, 2014,29(5):16-18. (in Chinese) |
| [20] | ( 孟庆贺. 我国包虫病的诊断和治疗进展[J]. 中国城乡企业卫生, 2014,29(5):16-18.) |
| [21] | Li YB, Yuan F, Li HW, et al. Application of fecal examination in investigation of infection source of echinococcosis[J]. J Ningxia Med Univ, 2010,32(1):147-150. (in Chinese) |
| [21] | ( 李燕兵, 袁芳, 李红卫, 等. 粪便检查在包虫病传染源调查中的应用[J]. 宁夏医科大学学报, 2010,32(1):147-150.) |
| [22] | Craig PS, Gasser RB, Parada L, et al. Diagnosis of canine echinococcosis: comparison of coproantigen and serum antibody tests with arecoline purgation in Uruguay[J]. Vet Parasitol, 1995,56(4):293-301. |
| [23] | Deplazes P, Alther P, Tanner I, et al. Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations[J]. J Parasitol, 1999,85(1):115-121. |
| [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) |
| [24] | ( 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] | Li T, Yang K. Application of isothermal amplification technology for pathogen detection in parasitic and other diseases[J]. Chin J Schisto Control, 2018,30(2):232-236. (in Chinese) |
| [25] | ( 李婷, 杨坤. 等温扩增技术在寄生虫及其他病原体检测中的应用[J]. 中国血吸虫病防治杂志, 2018,30(2):232-236.) |
| [26] | Ni XW. Development and application of LAMP method for the identification of Echinococcus spp. in dog faeces[D]. Beijing: Chinese Academy of Agricultural Sciences, 2012. (in Chinese) |
| [26] | ( 倪兴维. 犬棘球绦虫粪LAMP检测方法的建立和应用[D]. 北京: 中国农业科学院, 2012.) |
| [27] | Wassermann M, Mackenstedt U, Romig T. A loop-mediated isothermal amplification (LAMP) method for the identification of species within the Echinococcus granulosus complex[J]. Vet Parasitol, 2014,200(1/2):97-103. |
| [28] | Notomi T, Mori Y, Tomita N, et al. Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects[J]. J Microbiol, 2015,53(1):1-5. |
| [29] | Luo LH, Zhang B. Loop-mediated isothermal amplification method for the detection of infectious diseases[J]. Chin J Front Heal Quar, 2014,37(1):68-72. (in Chinese) |
| [29] | ( 罗力涵, 张波. 环介导等温扩增技术及其在传染性疾病检测中的应用[J]. 中国国境卫生检疫杂志, 2014,37(1):68-72.) |
/
| 〈 |
|
〉 |