Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in <25min.
Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in <25min.
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DOI：
10.1016/j.mimet.2016.11.010
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发表时间：
2017-01
影响因子：
2.2
通讯作者：
Hashsham SA
中科院分区：
文献类型：
--
作者：
Ahmad F;Stedtfeld RD;Waseem H;Williams MR;Cupples AM;Tiedje JM;Hashsham SA
我们正在报告一个最可能数的方法集成环介导的等温技术（MPN-LAMP）专注于革兰氏阴性大肠杆菌和革兰氏阳性粪肠球菌细菌细胞没有核酸提取。用LAMP法检测大肠杆菌uidA。coli和E.使用商业真实的时间PCR仪器，成功地直接在高达个位数浓度的细胞上进行粪便的PCR。细菌细胞、热处理的细菌细胞（95 °C，5分钟）及其纯化的基因组DNA模板的LAMP测定的阈值时间值是相似的，这意味着在63 °C下可以直接从细菌细胞实现扩增。细菌细胞的活力通过在LAMP测定中使用单叠氮丙啶与E.粪便为了检查其在微流体平台上的功能，还将靶向< 10 CFU细菌的MPN-LAMP测定转移到聚合物微芯片上，并通过低成本荧光成像系统进行监测。整个系统的信噪比（SNR）高达800，分析灵敏度< 10 CFU，阳性时间约为20分钟。MPN-LAMP检测的细胞浓度范围为105 CFU至< 10 CFU。来自LAMP测定的MPN值证实扩增来自< 10 CFU。本文描述的方法可直接应用于63 °C的细胞，消除了复杂的核酸提取步骤的要求，有利于开发灵敏、快速、低成本且可现场部署的系统。这种快速的MPN-LAMP方法有可能取代传统的MPN方法用于水生病原体。
We are reporting a most probable number approach integrated to loop mediated isothermal technique (MPN-LAMP) focusing on Gram-negative Escherichia coli and Gram-positive Enterococcus faecalis bacterial cells without nucleic acids extraction. LAMP assays for uidA from E. coli and gelE from E. faecalis were successfully performed directly on cells up to single digit concentration using a commercial real time PCR instrument. Threshold time values of LAMP assays of bacterial cells, heat treated bacterial cells (95 °C for 5 min), and their purified genomic DNA templates were similar, implying that amplification could be achieved directly from bacterial cells at 63 °C. Viability of bacterial cells was confirmed by using propidium monoazide in a LAMP assay with E. faecalis. To check its functionality on a microfluidic platform, MPN-LAMP assays targeting < 10 CFU of bacteria were also translated onto polymeric microchips and monitored by a low-cost fluorescence imaging system. The overall system provided signal-to-noise (SNR) ratios up to 800, analytical sensitivity of < 10 CFU, and time to positivity of about 20 min. MPN-LAMP assays were performed for cell concentrations in the range of 105 CFU to < 10 CFU. MPN values from LAMP assays confirmed that the amplifications were from < 10 CFU. The method described here, applicable directly on cells at 63 °C, eliminates the requirement of complex nucleic acids extraction steps, facilitating the development of sensitive, rapid, low-cost, and field-deployable systems. This rapid MPN-LAMP approach has the potential to replace conventional MPN method for waterborne pathogens.
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影响因子：
3.7
作者：
Myers FB;Henrikson RH;Bone JM;Lee LP
通讯作者：
Lee LP
DOI：
10.1073/pnas.0805309105
发表时间：
2008-12-09
影响因子：
11.1
作者：
Huang, Kerwyn Casey;Mukhopadhyay, Ranjan;Wingreen, Ned S.
通讯作者：
Wingreen, Ned S.
影响因子：
2.8
作者：
Ahmad, Farhan;Seyrig, Gregoire;Hashsham, Syed A.
通讯作者：
Hashsham, Syed A.
影响因子：
7.4
作者：
Atrazhev, Alexey;Manage, Dammika P.;Acker, Jason P.
通讯作者：
Acker, Jason P.
影响因子：
2.8
作者：
Mariella, Raymond, Jr.
通讯作者：
Mariella, Raymond, Jr.