Simultaneous detection of multiple viruses based on chemiluminescence and magnetic separation
Simultaneous detection of multiple viruses based on chemiluminescence and magnetic separation
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基于化学发光和磁分离的多种病毒同时检测
DOI：
10.1039/c6bm00527f
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发表时间：
2017-01-01
影响因子：
6.6
通讯作者：
Li, Zhiyang
中科院分区：
文献类型：
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作者：
Ali, Zeeshan;Wang, Jiuhai;Li, Zhiyang
基于核酸检测（NAT）的方法更灵敏和特异，并且优于酶免疫测定。已经设计了不同的基于NAT的协议来检测多种病原体，以降低检测的固有高成本。然而，这些测定不能可靠地一次检测大量病原体。本文提出了一种基于DNA杂交的化学发光检测方法，用于多种病原体的可靠检测。通过一步多重逆转录聚合酶链反应（RT-PCR）同时提取和扩增B型肝炎病毒（HBV）、C型肝炎病毒（HCV）和人免疫缺陷病毒（HIV-1 M组，在此称为HIV），实际证明了该想法。对影响化学发光信号的因素进行了详细的研究。针对HBV、HCV和HIV的探针通过仅捕获它们各自的靶序列而表现出更高的特异性。在HBV、HCV和HIV的单重检测方案中分别检测到10个病毒拷贝/mL血清。然而，当在多重检测试验中存在更高载量的其他病毒的情况下分析每种病毒的灵敏度时，该试验最终检测到每微升血清10个HBV拷贝、10个HCV拷贝和100个HIV拷贝。在核酸提取期间使用二氧化硅改性的磁性纳米颗粒和在化学发光步骤期间使用羧基包被的磁性纳米颗粒可以帮助将该系统修饰成用于高通量应用的自动化平台。
Nucleic acid testing (NAT) based methods are more sensitive and specific, and are preferred over enzyme immunoassays. Different NAT based protocols have been designed to detect multiple pathogens in order to reduce the inherent high cost for detection. However, these assays do not reliably detect a large number of pathogens at once. In this report, a DNA hybridization based chemiluminescence detection method has been proposed for reliable detection of multiple pathogens. The idea was practically demonstrated by carrying out simultaneous extraction and amplification of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV-1 group M, herein referred to as HIV) through the one step multiplex reverse transcription polymerase chain reaction (RT-PCR). A detailed study was conducted to optimize the factors which could affect the chemiluminescence signal. The probes for HBV, HCV, and HIV demonstrated higher specificity by only capturing their respective target sequence. 10 viral copies per mL of serum were detected in the monoplex detection protocol for HBV, HCV, and HIV, respectively. However, when the sensitivity of each virus was analyzed in the presence of higher loads of other viruses in the multiplex detection assay, the assay finally detected 10 HBV copies, 10 HCV copies, and 100 HIV copies per microliter of serum. The use of silica modified magnetic nanoparticles during nucleic acid extraction and carboxyl coated magnetic nanoparticles during the chemiluminescence step can help modify this system into an automated platform for high throughput applications.