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[Establishment and evaluation of the detection method of Cryptosporidium specific DNA fragment by recombinase aided isothermal amplification].

Abstract

OBJECTIVE

To establish a recombinase-aided isothermal amplification (RAA) assay for detection of Cryptosporidium.

METHODS

Based on Cryptosporidium-specific 18S rRNA selected as the target gene to be detected, and the primer sequences and fluorescent probes designed using the software Amplfix, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect 18S RNA target sequence-contained recombinant plasmids at various copies, genomic DNA of Cryptosporidium oocysts at various concentrations, and genomic DNA extracted from various numbers of Cryptosporidium oocysts to assess the sensitivity of the assay, and to detect genomic DNA extracted from Cryptosporidium oocysts, Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella and Shigella to determine the specificity of the assay.

RESULTS

A fluorescent RAA assay was successfully established, which was effective to amplify the specific 18S RNA gene fragments of Cryptosporidium within 20 min at 39 ℃. The lowest limits of the fluorescent RAA assay were 102 copies/μL for detection of 18S RNA target sequence-contained recombinant plasmids at various copies, 1 pg/μL for detection of genomic DNA of Cryptosporidium oocysts at various concentrations, and one Cryptosporidium oocyst/μL for detection of genomic DNA extracted from various numbers of Cryptosporidium oocysts, and the fluorescent RAA assay was all negative for detection of genomic DNA from G. lamblia cysts, S. japonicum eggs, A. lumbricoides eggs, C. sinensis eggs, Salmonella and Shigella.

CONCLUSIONS

A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of Cryptosporidium oocysts.

Authors+Show Affiliations

National Health Commission Key Laboratory of Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China. Public Health Research Center, Jiangnan University, China.National Health Commission Key Laboratory of Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China. Public Health Research Center, Jiangnan University, China.Jiangsu Qitian Gene Technology Co., Ltd, China.National Health Commission Key Laboratory of Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China. Public Health Research Center, Jiangnan University, China.Jiangsu Qitian Gene Technology Co., Ltd, China.National Health Commission Key Laboratory of Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China. Public Health Research Center, Jiangnan University, China.National Health Commission Key Laboratory of Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China. Public Health Research Center, Jiangnan University, China.

Pub Type(s)

Journal Article

Language

chi

PubMed ID

31612673

Citation

Bi-Xian, Ni, et al. "[Establishment and Evaluation of the Detection Method of Cryptosporidium Specific DNA Fragment By Recombinase Aided Isothermal Amplification]." Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi = Chinese Journal of Schistosomiasis Control, vol. 31, no. 4, 2019, pp. 388-392.
Bi-Xian N, Xiao-Min W, Yan-Hong L, et al. [Establishment and evaluation of the detection method of Cryptosporidium specific DNA fragment by recombinase aided isothermal amplification]. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2019;31(4):388-392.
Bi-Xian, N., Xiao-Min, W., Yan-Hong, L., Xiang-Zhen, X., Qing-Jie, Y., Jun, C., & Yang, D. (2019). [Establishment and evaluation of the detection method of Cryptosporidium specific DNA fragment by recombinase aided isothermal amplification]. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi = Chinese Journal of Schistosomiasis Control, 31(4), pp. 388-392. doi:10.16250/j.32.1374.2019168.
Bi-Xian N, et al. [Establishment and Evaluation of the Detection Method of Cryptosporidium Specific DNA Fragment By Recombinase Aided Isothermal Amplification]. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2019 Sep 19;31(4):388-392. PubMed PMID: 31612673.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Establishment and evaluation of the detection method of Cryptosporidium specific DNA fragment by recombinase aided isothermal amplification]. AU - Bi-Xian,Ni, AU - Xiao-Min,Wu, AU - Yan-Hong,Liu, AU - Xiang-Zhen,Xu, AU - Qing-Jie,Ying, AU - Jun,Cao, AU - Yang,Dai, PY - 2019/10/16/entrez PY - 2019/10/16/pubmed PY - 2019/10/18/medline KW - Cryptosporidium KW - Diagnostic efficiency KW - Fluorescent probe KW - Nucleic acid detection KW - Recombinase-aided isothermal amplification assay SP - 388 EP - 392 JF - Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control JO - Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi VL - 31 IS - 4 N2 - OBJECTIVE: To establish a recombinase-aided isothermal amplification (RAA) assay for detection of Cryptosporidium. METHODS: Based on Cryptosporidium-specific 18S rRNA selected as the target gene to be detected, and the primer sequences and fluorescent probes designed using the software Amplfix, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect 18S RNA target sequence-contained recombinant plasmids at various copies, genomic DNA of Cryptosporidium oocysts at various concentrations, and genomic DNA extracted from various numbers of Cryptosporidium oocysts to assess the sensitivity of the assay, and to detect genomic DNA extracted from Cryptosporidium oocysts, Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella and Shigella to determine the specificity of the assay. RESULTS: A fluorescent RAA assay was successfully established, which was effective to amplify the specific 18S RNA gene fragments of Cryptosporidium within 20 min at 39 ℃. The lowest limits of the fluorescent RAA assay were 102 copies/μL for detection of 18S RNA target sequence-contained recombinant plasmids at various copies, 1 pg/μL for detection of genomic DNA of Cryptosporidium oocysts at various concentrations, and one Cryptosporidium oocyst/μL for detection of genomic DNA extracted from various numbers of Cryptosporidium oocysts, and the fluorescent RAA assay was all negative for detection of genomic DNA from G. lamblia cysts, S. japonicum eggs, A. lumbricoides eggs, C. sinensis eggs, Salmonella and Shigella. CONCLUSIONS: A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of Cryptosporidium oocysts. SN - 1005-6661 UR - https://www.unboundmedicine.com/medline/citation/31612673/[Establishment_and_evaluation_of_the_detection_method_of_Cryptosporidium_specific_DNA_fragment_by_recombinase_aided_isothermal_amplification] DB - PRIME DP - Unbound Medicine ER -