Li Ting1, Liu Yan-Hong2, Zhao Song1, Xiong Chun-Rong1, Dong Xuan1, Zhang Jian-Feng1, Li Wei1, Ying Qing-Jie2, Yang Kun1. 1. 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. 2. Jiangsu Qitian Gene Technology Co., Ltd., China.
Abstract
OBJECTIVE: To develop a florescent recombinase-aided amplification (RAA) assay for rapid detection of Schistosoma japonicum-infected Oncomelania snails and explore the optimal method for treatment of snail samples. METHODS: Snail samples were divided into 3 groups, and each group consisted of 7 subgroups. There were 50 uninfected snails mixed with 1, 2, 3, 4, 5 and 10 infected snails in the 6 subgroups, respectively, and the remaining subgroup contained 100 uninfected snails mixed with 1 infected snails. DNA was extracted from snails in the three groups using a genomic DNA extraction kit following snail crushing and snail shells removal, crude nucleic acid extraction assay following snail crushing and snail shells removal, and crude nucleic acid extraction assay following direct snail crushing with snail shells preserved, and subjected to florescent RAA and PCR as says. The detection results were compared between the two assays. RESULTS: A florescent RAA assay was developed, which completed the detection of S. japonicum-infected snails at 39 ℃ within 30 min. Following DNA extraction from mass snail samples with a genomic DNA extraction kit following snail crushing and snail shells removal, the lowest detection limit of the florescent RAA assay was one infected snail mixed in 100 uninfected snails, while the lowest detection limit of PCR assay was one infected snail mixed in 50 uninfected snails. Following DNA extraction using crude nucleic acid extraction method following snail crushing and snail shells removal, the lowest detection limit of the florescent RAA assay was one infected snail mixed in 100 uninfected snails, while the lowest detection limit of PCR assay was 3 infected snails mixed in 50 uninfected snails. Following DNA extraction with a crude nucleic acid extraction assay following direct snail crushing with snail shells preserved, the lowest detection limit of the florescent RAA assay was 10 infected snails mixed in 50 uninfected snails, while the lowest detection limit of PCR assay was 10 infected snails mixed in 50 uninfected snails. CONCLUSIONS: A fluorescent RAA assay that is rapid to detect S. japonicum-infected snails in mass snail samples is successfully developed, which is fast, sensitive and easy to perform. Crude nucleic acid extraction following snail crushing and snail shells removal is the optimal method for the treatment of snail samples.
OBJECTIVE: To develop a florescent recombinase-aided amplification (RAA) assay for rapid detection of Schistosoma japonicum-infected Oncomelania snails and explore the optimal method for treatment of snail samples. METHODS: Snail samples were divided into 3 groups, and each group consisted of 7 subgroups. There were 50 uninfected snails mixed with 1, 2, 3, 4, 5 and 10 infected snails in the 6 subgroups, respectively, and the remaining subgroup contained 100 uninfected snails mixed with 1 infected snails. DNA was extracted from snails in the three groups using a genomic DNA extraction kit following snail crushing and snail shells removal, crude nucleic acid extraction assay following snail crushing and snail shells removal, and crude nucleic acid extraction assay following direct snail crushing with snail shells preserved, and subjected to florescent RAA and PCR as says. The detection results were compared between the two assays. RESULTS: A florescent RAA assay was developed, which completed the detection of S. japonicum-infected snails at 39 ℃ within 30 min. Following DNA extraction from mass snail samples with a genomic DNA extraction kit following snail crushing and snail shells removal, the lowest detection limit of the florescent RAA assay was one infected snail mixed in 100 uninfected snails, while the lowest detection limit of PCR assay was one infected snail mixed in 50 uninfected snails. Following DNA extraction using crude nucleic acid extraction method following snail crushing and snail shells removal, the lowest detection limit of the florescent RAA assay was one infected snail mixed in 100 uninfected snails, while the lowest detection limit of PCR assay was 3 infected snails mixed in 50 uninfected snails. Following DNA extraction with a crude nucleic acid extraction assay following direct snail crushing with snail shells preserved, the lowest detection limit of the florescent RAA assay was 10 infected snails mixed in 50 uninfected snails, while the lowest detection limit of PCR assay was 10 infected snails mixed in 50 uninfected snails. CONCLUSIONS: A fluorescent RAA assay that is rapid to detect S. japonicum-infected snails in mass snail samples is successfully developed, which is fast, sensitive and easy to perform. Crude nucleic acid extraction following snail crushing and snail shells removal is the optimal method for the treatment of snail samples.