2∣MATERIALS AND METHODS
2.1∣Materials
The RNA standards for SARS-CoV-2, SARS-CoV, Parainfluenza virus, Influenza virus A (FluA), and Influenza virus A (FluB) were synthesized by the Shanghai Institute of Measurement and Testing Technology. All primers, CRISPR RNA (crRNA), and the ssDNA reporter (FAM-TTATT-BHQ1) were synthesized by the Sangon Biotech Co. Ltd. Shanghai, China. EnGen® Lba Cas12a was purchased from New England Biolabs (Ipswich, MA, UK). The RNase inhibitor was purchased from Takara and the RPA amplification kit was purchased from Leshang Biotechnology (Wuxi) Co., LTD., China.
2.2∣Clinical samples
Nucleic acids are extracted from the total 60 nasopharyngeal swab samples, which collected in Renji Hospital. Among the 60 samples, according RT-PCR testing results, 30 nasopharyngeal swabs were positive for SARS-CoV-2, and 30 nasopharyngeal swabs were negative for SARS-CoV-2. Among those SARS-CoV-2 negative samples,
2 cases were diagnosed with influenza A, 5 cases with influenza B, and 2 with respiratory syncytial virus. The current study was approved by the ethics committees of Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital (Approval number: KY2020-192).
2.3∣VDAP detection
The RPA reaction was performed using the RPA kit according to the manufacturer’s instructions. In brief, RPA amplification mixture contained 30 μl of rehydration buffer, 2 μL of each primer (10 μM), 13 μL of ddH2O, and 2 μL of RNA. A total of 47 μL of the master mix was aliquoted into each reaction tube, and 3 μL magnesium acetate.
After optimization of reaction conditions, the reactions were conducted in a 37 ◦C incubator for 15 min. The information of primers is summarized in Table1. Every sample amplified product of the RPA reactions was subjected to VDAP. Specially, endogenous ribonuclease P RNA (RNase P) was set up as an internal reference control to monitor the process of clinical sample collection, preservation and transportation, as well as nucleic acid extraction to avoid false negative results misinterpretation The crRNAs were manually designed and the information of crRNAs was summarized in Table 2.
The VDAP was performed in a physically isolated room in order to avoid the aerosol contamination. The reaction system of the VDAP was 20 μL. The Cas12a reaction solution contained 1.2 µM crRNA, 0.4 µM Cas12a, 2 µM ssDNA reporter, 12 U RNase inhibitor, 2 μL 10 × NEB buffer 2.1, and 2 μL RPA amplified products. After 15 min of reaction at 37 ° C, a blue light gel imager and a smartphone were used to observe the fluorescence.