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.
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