Establishment of an automatic detection method for Middle East respira-tory syndrome coronavirus with an integrated microfluidic chip
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摘要:
目的 建立基于微流控芯片平台的中东呼吸综合征冠状病毒（MERS-CoV）一体化自动检测方法。 方法 制备MERS-CoV假病毒颗粒阳性参考品，设计针对病毒ORF1b基因区的引物和探针，制备基于实时荧光RT-PCR全流程的一体化集成微流控芯片，实现含样品裂解、核酸提取、核酸扩增和荧光检测的全自动检测。利用芯片内预包埋的内参基因对芯片系统性能进行监测，利用MERS-CoV假病毒阳性参考品对方法的灵敏度、特异性和重复性检测评价。 结果 微流控芯片中内参基因可以稳定扩增。自动化方法可检测原始样本中100拷贝（copies）的MERS-CoV假病毒，优于常规实时荧光RT-PCR（500 copies检出限）；100 copies假病毒重复测试6次CT值为（39.80 ± 0.53），变异系数为1.34 %。用自动化方法检测其他冠状病毒和人源基因均为阴性。 结论 建立的MERS-CoV微流控芯片一体化自动检测方法具有良好的稳定性、灵敏度和特异性，操作简便、快速，不受实验场地限制，可用于中东呼吸综合征现场即时检测。 -
关键词:
- 中东呼吸综合征冠状病毒(MERS-CoV) /
- 微流控芯片 /
- 荧光RT-PCR全流程快速检测
Abstract:Objective To develop an automatic detection method for Middle East respiratory syndrome coronavirus (MERS-CoV) based on integrated microfluidic chip platform. Methods The pseudovirus particle of MERS-CoV was prepared as positive reference and primers and probe were designed for the ORF1b gene region of the virus. Integrated microfluidic chips were created in accordance with the whole procedure of real-time reverse transcription polymerase chain reaction (RT-PCR), including viral lysis, nucleic acid extraction, amplification and real-time fluorescence detection. The performance of the chip was monitored by the internal reference gene embedded on the chip. The sensitivity, specificity and repeatability of the chip were evaluated using the MERS-CoV pseudovirus. Results The internal reference gene on the microfluidic chip could be amplified and detected stably. The automated detection was sensitive enough to detect as few as 100 copies of MERS-CoV in the original sample, which was more sensitive than normal real-time RT-PCR with the detection limitation of 500 copies. A cycle threshold (Ct) value of 39.80 ± 0.53, with a coefficient variation of 1.34%, was obtained when the test was repeated six times with 100 copies of pseudovirus. No signal was observed in automated tests for other coronaviruses and human genes. Conclusion The established automatic detection method with microfluidic chip is of good stability, sensitivity, and specificity and the method is fast and easy to operate, and is not limited by experimental sites. The method could be used for rapid detection of MERS-CoV in field investigation. -
表 1 MERS-CoV及内参基因的引物和探针序列
名称 序列 长度 基因位置 MERS-F TTCGATGTTGAGGGTGCTCAT 21 18266～18286 MERS-R TCACACCAGTTGAAAATCCTAATTG 25 18323～18347 MERS-P CCCGTAATGCATGTGGCACCAATGT 25 18291～18315 IPC-F AGTTGCAGTGTAACCGTCATGTA 22 IPC-R TCGACGAGACTCTGCTGTTAA 22 IPC-P CAGTAATCTGCGTCGCACGTGTGCA 20 
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