The Ethics Committee of The Second People’s Hospital of Fuyang approved this study. Existing samples collected during standard diagnostic tests were tested and analyzed by qRT-PCR, OSN-qRT-PCR, and ddPCR. No extra burden was posed to patients.
We retrospectively identified 24 hospitalized patients clinically diagnosed with COVID-19 between January 30, 2020, and February 17, 2020, in The Second People’s Hospital of Fuyang. Throat (n = 18) and anal (n = 4) swabs, sputum (n = 10), and blood (n = 2) samples were collected from the enrolled patients. All aspects of the study were performed according to national ethics regulations and approved by the Institutional Review Boards of China Center for Disease Control and Prevention (CDC). Written consent was obtained from patients or children’s parents.
SARS-CoV-2 pseudovirus preparation
The SARS-CoV-2 reference sequence was synthesized and cloned into a lentiviral vector and pseudovirus was prepared in 293T cells. The obtained pseudovirus contained RNA sequences of the ORF1ab and N genes in the lentiviral genome. The SARS-CoV-2 pseudovirus used in qRT-PCR and OSN-qRT-PCR was synthesized and processed by BDS company (DA’an, Guangzhou, China) at a RNA concentration of 2.0 × 104 copies/ml. The SARS-CoV-2 pseudovirus used in ddPCR was synthesized and processed by BioPerfectus Technologies Co. (Taizhou, China) at a RNA concentration of 1.5 × 105 copies/ml. The SARS-CoV-2 pseudoviral RNA was diluted with pseudovirus diluent (dilution ratio and method is shown in table 1), and SARS-CoV-2 pseudoviral RNA of the diluted samples S1, S2, S3, S4, S5, S6, S7, and S8 were extracted by using membrane adsorption kits (Di’an, Hangzhou, China).
Total RNA from throat and anal swabs, sputum, and blood samples from each patient was extracted from supernatants using Reagent of Nucleic Acid Extraction or Purification (Di’an, Hangzhou, China) following the manufacturer’s instructions. SARS-CoV-2 nucleic acid detection was mainly targeted at the two-segment conserved gene sequence of its genome, located at ORF1ab and N.
Reaction components of the ddPCR assay kit (BioPerfectus) included 5 µl of Supermix, 2 µl of reverse transcriptase, 1 µl of 300 mM DTT, 5 µl of SARS-CoV-2 reaction solution, and 7 µl template. All procedures followed the manufacturer’s instructions for the QX200 Droplet Digital PCR System using Supermix for the probe (no dUTP) (Bio-Rad, Hercules, USA). 20 µl of each reaction mix was converted to droplets with the QX200 droplet generator. Droplet-partitioned samples were then transferred to a 96-well plate, sealed, and cycled in a C1000 Touch Thermal Cycler (Bio-Rad) under the following cycling protocol: 50°C for 60 min and 95°C for 10 min, followed by 40 cycles of 95°C for 30 s and 56°C for 1 min, then 98°C for 10 min and 4°C hold. FAM (ORF1ab) and HEX (N) channels were selected to detect SARS-CoV-2. The cycled plate was then transferred and FAM and HEX channels read using the QX200 reader. Each run contained positive and negative controls. Samples were only considered positive when both FAM and HEX channels had signals.
Reaction components of the OSN-qRT-PCR assay kit (Sansure, Changsha, China) included 20 µl of template, 26 µl of reaction buffer, and 4 µl of the enzyme mixture. After vortexing and centrifugation, the reaction tube was transferred to the LightCycler 480 II Real-Time PCR System (Roche, Basel, Switzerland). The OSN-qRT-PCR amplification reaction contained the following steps: 50°C for 30 min, 95°C for 1 min, 20 cycles at 95°C for 30 s, 70°C for 40 s, and 72°C for 40s, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 25°C for 10 s of instrument cooling. FAM (ORF1ab) and ROX (N) channels were selected to detect SARS-CoV-2, and the VIC channel was chosen to detect the reference gene (human ABL1). Each run contained positive and negative controls. FAM, HEX, and VIC channels all showed typical S-shaped amplification curves. The result was considered valid when the cycle threshold (Ct) value of the reference gene was ≤37. The result was considered positive when the Ct values of both target genes were ≤35 and negative when they were both >35. If only one of the target genes had a Ct value ≤35 and the other was >35, it was interpreted as a single-gene positive.
The qRT-PCR kit (DaAn Gene; Guangzhou, China) included 17 µl of SARS-CoV-2 NC reaction solution A, 3 µl of NC reaction solution B, and 5 µl of template. After vortexing and centrifugation, the reaction tube was transferred to the LightCycler 480 II Real-Time PCR System (Roche). The qRT-PCR amplification reaction contained the following steps: 50°C for 15 min, 95°C for 15 min, 45 cycles at 94°C for 15 s, and 55°C for 45 s. FAM (N) and VIC (ORF1ab) channels were selected to detect SARS-CoV-2, and the CY5 channel was chosen to detect the reference gene (human ABL1). The result was considered valid when the Ct value of the reference gene was ≤37. The result was considered positive when the Ct values of both target genes (ORF1ab and N) were ≤37 and were considered negative when they were both >40. If only one of the target genes had a Ct value fall in the gray zone (37–40), it was retested. If the repeated result was positive for only one of two targets genes, it was interpreted as positive.
Dynamic range and LoD of OSN-qRT-PCR, ddPCR, and qRT-PCR
To evaluate the dynamic range and consistency of OSN-qRT-PCR, ddPCR, and qRT-PCR, we first ran a serial dilution of the linear RNA standard for each assay. To determine the LoD, the lower concentration RNA standards (including S3–S8) were analyzed 14 times. The LoD was calculated by Probit regression analysis with a 95% repeatable probability.
Data statistical analysis
Analysis of the ddPCR data was performed with Quanta Soft Analysis Software v1.7.4 to calculate the concentration of the target. Plots of linear regression were conducted with GraphPad Prism 7.0, and Probit analysis for LoD was conducted with MedCalc software v19.2.1. Bland-Altman analysis of qRT-PCR, OSN-qRT-PCR, and ddPCR results for patient samples was evaluated by SPSS 23.0 statistical software.