Spectrophotometric, colorimetric and visually detection of Pseudomonas aeruginosa ETA gene based gold nanoparticles DNA probe and endonuclease enzyme
Spectrophotometric, colorimetric and visually detection of Pseudomonas aeruginosa ETA gene based gold nanoparticles DNA probe and endonuclease enzyme
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DOI：
10.1016/j.saa.2018.03.056
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发表时间：
2018-06-15
影响因子：
4.4
通讯作者：
Yaghmaei, Parichehreh
中科院分区：
文献类型：
--
作者：
Amini, Bahram;Kamali, Mehdi;Yaghmaei, Parichehreh
比色法DNA检测优于临床分子诊断的其他方法，因为它不需要昂贵的设备。本研究采用纳米金颗粒（gold nanopartrides，GNP）-核酸内切酶比色法检测铜绿假单胞菌ETA基因。首先，设计了铜绿假单胞菌外毒素A（ETA）基因的引物和探针，并通过PCR方法进行了特异性检测。采用柠檬酸盐还原法合成了GNP，并将其与探针偶联制备了新型纳米生物传感器。接着，将提取的细菌的靶DNA加入GNP-探针复合物中以检查其用于铜绿假单胞菌ETA基因诊断的功效。当GNP-探针-靶DNA切割成BamHI核酸内切酶的小片段时，由于GNP和缩短的DNA之间的静电相互作用减弱，可见吸光度降低。加入核酸内切酶后，530 - 562 nm的吸收峰发生右移。使用UV-VIS吸收光谱法测量，其指示铜绿假单胞菌ETA基因的存在。在存在不同浓度的铜绿假单胞菌靶DNA的情况下测定灵敏度。结果表明，在10-50 ng·mL ~（-1）范围内，吸光度值与目标DNA浓度呈良好的线性关系（R：0.9850），检测限为9.899 ng·mL ~（-1）。因此，与其他细菌相比，确定了检测铜绿假单胞菌的新方法的专属性。此外，设计的检测试剂盒定量用于检测真实的样本中10（3）至10（8）CFU mL（-1）的铜绿假单胞菌ETA基因，检测限为320 CFU（C）2018 Elsevier B. V.保留所有权利。
Colorimetric DNA detection is preferred over other methods for clinical molecular diagnosis because it does not require expensive equipment. In the present study, the colorimetric method based on gold nanopartides (GNPs) and endonuclease enzyme was used for the detection of P. aeruginosa ETA gene. Firstly, the primers and probe for P. aeruginosa exotoxin A (ETA) gene were designed and checked for specificity by the PCR method. Then, GNPs were synthesized using the citrate reduction method and conjugated with the prepared probe to develop the new nano-biosensor. Next, the extracted target DNA of the bacteria was added to GNP-probe complex to check its efficacy for P. aeruginosa ETA gene diagnosis. A decrease in absorbance was seen when GNP-probe-target DNA cleaved into the small fragments of BamHI endonuclease due to the weakened electrostatic interaction between GNPs and the shortened DNA. The right shift of the absorbance peak from 530 to 562 nm occurred after adding the endonuclease. It was measured using a UV-VIS absorption spectroscopy that indicates the existence of the P. aeruginosa ETA gene. Sensitivity was determined in the presence of different concentrations of target DNA of P. aeruginosa. The results obtained from the optimized conditions showed that the absorbance value has linear correlation with concentration of target DNA (R: 0.9850) in the range of 10-50 ng mL(-1) with the limit detection of 9.899 ng mL(-1). Thus, the specificity of the new method for detection of P. aeruginosa was established in comparison with other bacteria. Additionally, the designed assay was quantitatively applied to detect the P. aeruginosa ETA gene from 10(3) to 10(8) CFU mL(-1) in real samples with a detection limit of 320 CFU (C) 2018 Elsevier B.V. All rights reserved.