Case definitions. In accordance with the latest guidelines of Diagnosis and Treatment of Novel Coronavirus Pneumonia (Trial Version 7) from the National Health Commission & State Administration of Traditional Chinese Medicine of China (available at http://en.nhc.gov.cn/2020-03/29/c_78469.htm), a suspected case was defined as a person with any of the following epidemiological history plus any two of the following clinical manifestations, or all three clinical manifestations if there was no clear epidemiological history. 1) Epidemiological history: within 14 days prior to the onset of illness, a) traveled to or took residence, or b) had contact with individuals who had fever or respiratory symptoms, in Wuhan and geographical proximities or communities where cases had been reported; c) had contact with any laboratory confirmed cases; d) was part of a cluster of two or more cases with fever and/or respiratory symptoms. 2) Clinical manifestations: a) fever and/or respiratory symptoms; b) defined imaging characteristic of SARS-CoV-2 pneumonia; c) normal or decreased white blood cell count and/or lymphocyte count in the early stages of illness onset.
Confirmed cases were defined as suspected cases with one of the following etiological evidences: (1) a positive RT-qPCR test for SARS-CoV-2; (2) viral gene sequence highly homologous to SARS-CoV-2; (3) SARS-CoV-2 specific IgM and IgG antibodies detectable in serum, IgG detectable or reaching a titration of at least a four-fold increase during convalescence compared with the acute phase. Negative cases were defined as suspected cases with two consecutive negative nucleic acid tests taken at least 24-hour apart and that SARS-CoV-2 specific IgM and IgG antibodies were negative after 7 days from illness onset.
Clinical samples. Respiratory samples (n=52) were collected from suspected cases at the Shenzhen Third People’s Hospital and a compulsory quarantine facility in Shenzhen. Specimen types included throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprising clinical manifestationor PCR-confirmed (n=26) and -negative (n=26) cases.
Nucleic acid amplification. All six kits used the ORF1ab gene and the N gene as targets (ORF1ab only for Kit D) and were performed in accordance with the manufacturer’s instructions (Table 1). Briefly, in the automated CPA kit (Kit A), swab samples were directly applied to cartridges preloaded with reagents for nucleic acid purification, elution buffer and CPA reaction master mix, in which RNA were automatically purified, extracted and amplified. Fluorescence-labeled probes bind specifically to the amplified RNA targets to produce a fluorescent signal detectable by the instrument in real time for the determination of test results. For the RT-qPCR kits (Kits B–F), RNA was manually extracted with a High Pure Viral Nucleic Acid kit (Roche Diagnostics, Mannheim, Germany). For Kit B, the PCR was carried out in a 25 μL reaction volume containing 5 μL isolated DNA as template, 4 μL primer and probe mix (ORF1ab/N) and 4 μL Premix ExTaq. Amplification began with one cycle of reverse transcription at 50℃ for 10 min and a pre-denaturation step at 95°C for 5 min, followed by 40 cycles of 95°C for 10s, 55°C for 40s. For Kit C, the PCR was carried out in a 25 μL reaction volume containing 5 μL viral cDNA as template, 19 μL RT-qPCR buffer and 1 μL enzyme mix. Amplification began with one cycle of reverse transcription at 42℃ for 15 min and a pre-denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 5s, 60°C for 45s. For Kit D, the 30 μL RT-qPCR reaction mixture contained 10 μL viral cDNA as template, 18.5 μL RT-qPCR buffer and 1.5 μL enzyme mix. Amplification began with one cycle of reverse transcription at 50℃ for 20 min and a pre-denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 15s, 60°C for 30s. For Kit E, the 20 μL RT-qPCR reaction mixture contained 4 μL viral cDNA template, 18.5 μL RT-qPCR buffer and 1.5 μL enzyme mix. Amplification began with one cycle of reverse transcription at 55℃ for 15 min and a pre-denaturation step at 95°C for 30 min, followed by 45 cycles of 95°C for 10s, 60°C for 30s. For Kit F, the PCR was carried out in a 25 μL reaction volume containing 5 μL viral cDNA template, 17 μL RT-qPCR Buffer A and 3 μL RT-qPCR Buffer B. Amplification began with one cycle of reverse transcription at 50℃ for 15 min and a pre-denaturation step at 95°C for 15 min, followed by 45 cycles of 94°C for 15s, 55°C for 45s. The assays were performed on an ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA), Fluorescent signal was detected at the end of the extension step of each cycle. The results were interpreted by CT values generated using specific cut-offs for each kit (Table 1).
Table 1. Parameters for each of the six nucleic acid amplification kits evaluated
Kit
|
Target
|
Volume (uL)
|
|
Results interpretation
|
LOD (copies/mL)
|
Ref
|
sample
|
RNA
|
|
Positive
|
Negative
|
A
|
ORF 1ab, N
|
500
|
/
|
|
Tt a≤40
|
No Tt value
|
1000
|
Y
|
B
|
ORF 1ab, N
|
/
|
5
|
|
Ct≤38
|
No Ct value or Ct>38
|
1000
|
N
|
C
|
ORF 1ab, N
|
/
|
5
|
|
Ct≤35
|
Ct>38
|
500
|
N
|
D
|
ORF 1ab
|
/
|
10
|
|
Ct≤32
|
FAM:No Ct value VIC/HEX:Ct≤32
|
100
|
Y
|
|
|
E
|
ORF 1ab, N
|
/
|
4
|
|
Ct<38
|
No Ct value or Ct=45
|
300
|
N
|
F
|
ORF 1ab, N
|
/
|
5
|
|
Ct≤40
|
No Ct value or Ct>40
|
500
|
Y
|
a Tt value is unique to the CPA kit (Kit A) and represents the time when fluorescence value exceeds the threshold line.
Analytical performance of six nucleic acid amplification kits. Analytical sensitivity and specificity, the positive and negative predictive values (PPV and NPV) and the accuracy for each kit were assessed using clinical diagnostic reports as the reference standard.
Reproducibility of RT-qPCR kits. Reproducibility for the RT-qPCR kits was evaluated using a positive sample verified by all kits, measured in triplicate by three different operators. The means, standard deviations (SD) and the coefficients of variation (CV) were calculated.
Statistical analysis. Statistical analyses were performed using SPSS software version 21.0 (IBM Corp., Armonk, NY). Confidence intervals (95%) were computed using the Wilson Score method. The agreement between nucleic acid amplification kits and standard diagnosis methods were compared using the McNemar-Bowker test.
Clinical application. Additional throat swabs (n=200) from close contacts of confirmed cases were obtained from the compulsory quarantine facility to further compare Kit A with Kits B and F (approved and authorized by the National Medical Products Administration for clinical use) . Agreement between three kits was assessed.