Department of Medical Microbiology & Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
Biosens Bioelectron. 2012 Oct-Dec;38(1):151-6. doi: 10.1016/j.bios.2012.05.019. Epub 2012 May 24.
In the field of diagnostics, molecular amplification targeting unique genetic signature sequences has been widely used for rapid identification of infectious agents, which significantly aids physicians in determining the choice of treatment as well as providing important epidemiological data for surveillance and disease control assessment. We report the development of a rapid nucleic acid lateral flow biosensor (NALFB) in a dry-reagent strip format for the sequence-specific detection of single-stranded polymerase chain reaction (PCR) amplicons at ambient temperature (22-25°C). The NALFB was developed in combination with a linear-after-the-exponential PCR assay and the applicability of this biosensor was demonstrated through detection of the cholera toxin gene from diarrheal-causing toxigenic Vibrio cholerae. Amplification using the advanced asymmetric PCR boosts the production of fluorescein-labeled single-stranded amplicons, allowing capture probes immobilized on the NALFB to hybridize specifically with complementary targets in situ on the strip. Subsequent visual formation of red lines is achieved through the binding of conjugated gold nanoparticles to the fluorescein label of the captured amplicons. The visual detection limit observed with synthetic target DNA was 0.3 ng and 1 pg with pure genomic DNA. Evaluation of the NALFB with 164 strains of V. cholerae and non-V. cholerae bacteria recorded 100% for both sensitivity and specificity. The whole procedure of the low-cost NALFB, which is performed at ambient temperature, eliminates the need for preheated buffers or additional equipment, greatly simplifying the protocol for sequence-specific PCR amplicon analysis.
在诊断领域，针对独特遗传特征序列的分子扩增已被广泛用于快速鉴定传染病原体，这极大地帮助医生确定治疗选择，并提供重要的流行病学数据以进行监测和疾病控制评估。我们报告了一种快速核酸侧向流动生物传感器（NALFB）的开发，该传感器采用干燥试剂条格式，可在环境温度（22-25°C）下针对单链聚合酶链反应（PCR）扩增子进行序列特异性检测。NALFB 与线性后指数 PCR 测定法相结合开发，通过检测引起腹泻的产毒霍乱弧菌中的霍乱毒素基因，证明了该生物传感器的适用性。使用先进的不对称 PCR 扩增可提高荧光标记的单链扩增子的产量，使固定在 NALFB 上的捕获探针能够在条带上原位与互补靶标特异性杂交。随后，通过共轭金纳米粒子与捕获的扩增子上的荧光标记物结合，形成红色线。用合成靶 DNA 观察到的视觉检测限为 0.3 ng，用纯基因组 DNA 观察到的视觉检测限为 1 pg。对 164 株霍乱弧菌和非霍乱弧菌细菌的 NALFB 评估显示，敏感性和特异性均为 100%。这种低成本的 NALFB 整个过程在环境温度下进行，无需预热缓冲液或额外设备，极大地简化了针对特定序列的 PCR 扩增子分析的方案。
