Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications.
Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications.
复制标题
用于微阵列应用的NASBA扩增的TMRNA分子的荧光标记。
DOI：
10.1186/1472-6750-9-45
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发表时间：
2009-05-15
影响因子：
3.5
通讯作者：
Kurg A
中科院分区：
文献类型：
--
作者：
Scheler O;Glynn B;Parkel S;Palta P;Toome K;Kaplinski L;Remm M;Maher M;Kurg A
本文提出了一种新的微生物诊断方法，该方法将核酸序列扩增（NASBA）的敏感性与微阵列技术的高信息量相结合，用于检测细菌tmRNA分子。NASBA协议被修改为包括在NASBA反应中被纳入新生RNA的氨基丙烯基utp （aaUTP）分子。随后用荧光染料进行扩增后标记，并使用微阵列技术测量tmRNA杂交信号强度。需要对标记的NASBA方案进行重大优化，以保持反应所需的灵敏度。对两种不同的aaUTP盐进行了评价，并确定了两种盐的最佳最终浓度。在NASBA反应中，最终浓度为2 mM的aaUTP Li-salt产生的微阵列信号总体上最高，是1 mM aaUTP Na-salt最强信号的两倍。我们已经成功地展示了NASBA扩增技术与基于微阵列的杂交检测的有效结合。该方法适用于微生物诊断的许多不同领域，包括环境监测、生物威胁检测、工业过程监测和临床微生物学。
Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology.
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影响因子：
14.9
作者：
't Hoen, PAC;de Kort, F;den Dunnen, JT
通讯作者：
den Dunnen, JT
影响因子：
4.2
作者：
Schonhuber, W;Le Bourhis, G;Kulakauskas, S
通讯作者：
Kulakauskas, S
DOI：
10.1016/j.bbrc.2006.07.039
发表时间：
2006-09-08
影响因子：
3.1
作者：
Gill, Pooria;Ramezani, Reihaneh;Tehrani, Hossein A.
通讯作者：
Tehrani, Hossein A.
影响因子：
2.7
作者：
Richter, A;Schwager, C;Muckenthaler, M
通讯作者：
Muckenthaler, M
影响因子：
2.7
作者：
Hegde, P;Qi, R;Quackenbush, J
通讯作者：
Quackenbush, J