In situ, amplification-free double-stranded mutation detection at 60 copies/ml with thousand-fold wild type in urine.
In situ, amplification-free double-stranded mutation detection at 60 copies/ml with thousand-fold wild type in urine.
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DOI：
10.1016/j.bios.2018.07.062
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发表时间：
2018-11-15
影响因子：
12.6
通讯作者：
Shih WY
中科院分区：
文献类型：
--
作者：
Kirimli C;Lin S;Su YH;Shih WH;Shih WY
我们已经研究了使用压电板传感器（PEP）在10− 19 M- 10−16 M浓度范围内的尿液中的无扩增原位双链突变检测。在具有两个温度区的闭环流动中进行检测。95°C高温区用作样品加载和DNA去杂交的储库。将加热的尿液以4 ml/min的流速流过浸没在室温水浴中的1 m长的管冷却，以在进行检测所需的检测温度下到达检测池。以B肝炎病毒双突变（HBVDM）和KRAS G12 V点突变作为模型双突变，结果表明，与相同浓度下的单链突变检测相比，PEPS能够在低至10− 19 M的浓度下以70%或更高的检测效率检测双链HBVDM和KRAS，这通过以下原位荧光报告微球（FRM）检测以及结合于PEPS表面上捕获的突变体的FRM的显微镜可视化来验证。此外，在HBVDM为250倍野生型和KRAS为1000倍野生型的背景下，在10−19 M - 10−16 M下证明了相同的双链突变检测效力。还证明了在尿液中10−19 M - 10−16 M的SW 480 DNA片段处检测到KRAS突变。
We have investigated amplification-free in situ double-stranded mutation detection in urine in the concentration range 10−19M – 10−16 M using piezoelectric plate sensors (PEPs). The detection was carried out in a close-loop flow with two temperature zones. The 95°C high-temperature zone served as the reservoir where the sample was loaded and DNA de-hybridized. The heated urine was cooled flowing through a 1 m long tubing immersed in room-temperature water bath at a flow rate of 4 ml/min to reach the detection cell at the desire detection temperature for the detection to take place. With hepatitis B virus double mutation (HBVDM) and KRAS G12V point mutation as model double mutations, it is shown that PEPS was able to detect double-stranded HBVDM and KRAS with 70% detection efficiency or better at concentration as low as 10−19M against single-stranded mutation detection at the same concentrations, which was validated by the following in situ fluorescent reporter microspheres (FRMs) detection as well as microscopic visualization of the FRMs bound to the captured mutant on the PEPS surface. Furthermore, the same double-stranded mutation detection efficacy was demonstrated at 10−19 M – 10−16 M in a background of 250-fold wildtype for HBVDM and 1000-fold wildtype for KRAS. Also demonstrated was detection of KRAS mutation at 10−19 M – 10−16 M of SW480 DNA fragments in urine.
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DOI：
10.1039/c5an02048d
发表时间：
2016-02-21
期刊：
The Analyst
影响因子：
--
作者：
Kirimli CE;Shih WH;Shih WY
通讯作者：
Shih WY
影响因子：
16.6
作者：
Kurkina, Tetiana;Vlandas, Alexis;Balasubramanian, Kannan
通讯作者：
Balasubramanian, Kannan
影响因子：
7.4
作者：
Loaiza, Oscar A.;Campuzano, Susana;Pingarron, Jose M.
通讯作者：
Pingarron, Jose M.
影响因子：
16.6
作者：
Hammond, David M.;Manetto, Antonio;Carell, Thomas
通讯作者：
Carell, Thomas
影响因子：
15
作者：
He, L;Musick, MD;Keating, CD
通讯作者：
Keating, CD