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.