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微生物学通报

包装饮用水中铜绿假单胞菌快速检测试剂盒的研制与评价
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国家重点研发计划(2018YFC1604201);广东省自然科学基金(2017A030310090);肇庆市引进西江创新团队项目(肇人才领2018-8)


Development and evaluation of loop-mediated isothermal amplification-based kit for rapid detection of Pseudomonas aeruginosa in packaged drinking water
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    摘要:

    【背景】在包装饮用水企业生产活动中,铜绿假单胞菌是被重点监测的致病菌之一。随着分子检测相关技术的不断发展,研制用于包装饮用水中铜绿假单胞菌简便、高效的快速检测产品至关重要。【目的】评价基于环介导恒温扩增(loop-mediated isothermal amplification,LAMP)技术的铜绿假单胞菌快速检测试剂盒在包装饮用水铜绿假单胞菌检测中的实效性。【方法】优化该LAMP反应体系,反应试剂采用冻干工艺,确定试剂盒组成,并评价其特异性、灵敏度、重复性、保质期等性能指标。【结果】铜绿假单胞菌标准菌株和分离菌株均检测为阳性,非铜绿假单胞菌标准菌株和分离菌株均检测为阴性,未发现有交叉反应;试剂盒最低检验限为18 CFU/mL;该试剂盒的特异性、灵敏度及准确度与传统方法相比具有较高的一致性;批内、批间检测重复率均为100%,可在4 °C保存12个月以上,并且可在42 °C环境中储存72 h以上。【结论】该试剂盒性能好,检测结果稳定、可靠,适用于包装饮用水中铜绿假单胞菌的快速检测。

    Abstract:

    [Background] Pseudomonas aeruginosa is a major pathogen that needed to be intensively monitored in the production activities by packaged drinking water enterprises. With continuous development of the molecular detection technology, it is very important to develop simple and efficient detection products to detect P. aeruginosa in packaged drinking water. [Objective] To develop loop-mediated isothermal amplification (LAMP) based kit for rapid detecting P. aeruginosa and evaluate its detecting efficacy in packaged drinking water. [Methods] We optimized the LAMP reaction system and confirmed the components of the kit. The main reaction reagents were made into powder pattern by freeze drying technology. Then the specificity, sensitivity, repeatability, shelf life and other performance indexes of the kit were evaluated. [Results] All P. aeruginosa standard strains and isolated strains were detected positive, while all non-P. aeruginosa standard strains and isolated strains were detected negative, showing that the detection had no cross reaction. The minimum test limit of the kit was calculated to 18 CFU/mL, and detection specificity, accuracy and sensitivity of the kit were preeminent. The detection result was highly consistent with that obtained by conventional microbial detection method. The intra-batch and inter-batch testing repetition rates were all 100%. The kit could be preserved for more than 12 months at 4 °C, and its efficacy was mostly maintained for more than 72 h at 42 °C. [Conclusion] The kit has good performance with stable and reliable test results which make it applicable for rapid detection of P. aeruginosa in packaged drinking water.

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周杨,万强,蔡芷荷,卢勉飞,曲晓莹,吴清平,李健顺. 包装饮用水中铜绿假单胞菌快速检测试剂盒的研制与评价[J]. 微生物学通报, 2020, 47(6): 1982-1992

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  • 在线发布日期: 2020-06-01
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