RESUMEN
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.
RESUMEN
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.
RESUMEN
Silicon quantum dot has become an attractive nanomaterial due to their excellent biocompatibility and optical performance. However, poor water-solubility of the traditional silicon quantum dot limits its wide application. In this study, we reported the synthesis of water-soluble silicon quantum dots with imidazole groups by using hydrothermal method, in which N-trimethysilylimidazole was used as a precursor of silicon. Compared with sodium borohydride, ascorbic acid, bovine serum protein, cysteine and citric acid, the as-prepared silicon quantum dots offered the strongest fluorescence intensity when sodium citrate was used as the reducing agent and stabilizer for the synthesis. The reaction could complete within 2 h at 220℃. The obtained silicon quantum dots showed good water-solubility with an average particle size of 2. 6 nm, and the result of infrared spectroscopic analysis verified the existence of free imidazole groups on the surface. By means of the investigation of the fluorescence quenching behavior of copper ions towards the silicon quantum dots at different temperatures, we found that the degree of fluorescence quenching increased with the increase of temperature. There results proved that the fluorescence decrease belongs to static quenching. Namely, the interaction of Cu2+ with imidazole groups on the surface of silicon quantum dots formed stable complex. In addition, the resonance light scattering analysis also showed that the fluorescence quenching process was accompanied by the agglomeration of particles. Based on the fluorescence quenching behavior of silicon quantum dots, we established a method for the fluorescent detection of Cu2+. When the concentration of Cu2+was in the range of 0. 04-2400 μmol/L, the fluorescence intensity would linearly decrease with the increase of Cu2+ concentration, and the detection limit (S/N=3) reached 1. 29×10-8 mol/L. The method provided high sensitivity, selectivity and reproducibility, and was successfully applied to the determination of trace copper in fruits and vegetables.