A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips
A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips
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DOI：
10.1007/s10544-011-9562-2
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发表时间：
2011-10-01
影响因子：
2.8
通讯作者：
Hashsham, Syed A.
中科院分区：
文献类型：
--
作者：
Ahmad, Farhan;Seyrig, Gregoire;Hashsham, Syed A.
对于疾病的早期诊断和治疗，尤其是在资源有限的环境中，需要快速、灵敏和低成本的病原体诊断系统。本研究报告了一种基于电荷耦合器件(CCD)的低成本荧光成像系统，用于通过在一次性微芯片中进行等温基因扩增来快速检测水媒病原体。通过优化增益、偏移量和曝光时间来评价这种单色CCD相机的荧光成像能力。该成像系统在环烯烃聚合物(COP)芯片上验证了12种主要水媒病原体的毒力基因，使用Syto-82染料和实时荧光环路介导的等温扩增，这里称为microRT(F)-LAMP。将MicroRT(F)-LAMP检测的信噪比(SNR)和阈值时间(TT)与商用实时聚合酶链式反应(PCR)仪的检测结果进行了比较。使用5 S对10(5)起始DNA拷贝的MicroRT(F)-LAMP分析的CCD光照射，与商业实时荧光PCR仪相比，SNR提高了8倍，TT降低了9.8分钟。此外，MicroRT(F)-LAMP对空肠弯曲菌0414基因的单拷贝水平敏感性达到了19min，是商用实时荧光PCR仪的一半。由于对曝光时间的控制和CCD的宽视场成像能力，这种低成本的荧光成像系统具有快速、并行检测高通量微流控芯片中病原微生物的潜力。
Rapid, sensitive, and low-cost pathogen diagnostic systems are needed for early disease diagnosis and treatment, especially in resource-limited settings. This study reports a low-cost charge-coupled device (CCD)-based fluorescence imaging system for rapid detection of waterborne pathogens by isothermal gene amplification in disposable microchips. Fluorescence imaging capability of this monochromatic CCD camera is evaluated by optimizing the gain, offset, and exposure time. This imaging system is validated for 12 virulence genes of major waterborne pathogens on cyclic olefin polymer (COP) microchips, using SYTO-82 dye and real time fluorescence loop-mediated isothermal amplification referred here as microRT(f)-LAMP. Signal-to-noise ratio (SNR) and threshold time (Tt) of microRT(f)-LAMP assays are compared with those from a commercial real-time polymerase chain reaction (PCR) instrument. Applying a CCD exposure of 5 s to 10(5) starting DNA copies of microRT(f)-LAMP assays increases the SNR by 8-fold and reduces the Tt by 9.8 min in comparison to a commercial real-time PCR instrument. Additionally, single copy level sensitivity for Campylobacter jejuni 0414 gene is obtained for microRT(f)-LAMP with a Tt of 19 min, which is half the time of the commercial real-time PCR instrument. Due to the control over the exposure time and the wide field imaging capability of CCD, this low-cost fluorescence imaging system has the potential for rapid and parallel detection of pathogenic microorganisms in high throughput microfluidic chips.