A total of 506 nasopharyngeal swabs (273 samples in Hangzhou Customs and, 233 samples in Zhoushan Customs) were collected from visitors entering China between January 1, 2021, and January 25, 2021. The sample collection and preservation were performed according to the Guide of Laboratory Techniques for testing novel coronavirus infected pneumonia released by the National Health Commission of the People’s Republic of China. After collection, the samples were inactivated at 56 °C for 30 min and stored at -80 °C. This study was approved by the institutional review committees of Hangzhou and Zhoushan Customs and was conducted as per the National Code of Ethics.
Nucleic acid extraction was performed using the existing automated nucleic acid extractor that is routinely used at customs for detecting SARS-CoV-2 RNA. The specific nucleic acid extraction methods used at customs were summarized in Table 1. Total DNA/RNA was extracted from the samples using the methods outlined in Table 1, according to the manufacturers’ instructions, and stored at -80 °C until use.
Manufacturer Instrument model Reagent Extraction duration (min) Institution Tianlong GeneRotex 96 Nucleic acid extraction kit 24 Hangzhou customs Tianlong NP968 Nucleic acid extraction kit 15 Zhoushan customs Tiangen TGuide S32 Total RNA extraction kit 18 Zhoushan customs Bioperfectus
Viral nucleic acid extraction kit
Note: Detection of SARS-CoV-2 viral RNA in the clinical samples using RT-RAA kits.
Abbreviation: RT-RAA=real-time reverse transcription recombinase-aided amplification.
Table 1. Nucleic acid extraction methods used at the customs.
SARS-CoV-2 RNA was detected from the clinical samples using the COVID-19 RT-RAA kit, according to the methods described in our previous report (10-11). Positive controls (recombinant plasmids) and negative controls (DNase-free water) were included in each run to ensure the reliability of the experimental results. The 6-carboxy-fluoescein (FAM) channel was used to detect the amplification of the target gene, whereas the Hexachloro fluorescein (HEX) channel was used to detect the amplification of the internal control gene. The results were considered to be positive when both channels were positive or when the FAM channel was positive, and the HEX channel was negative. When the FAM channel was negative, but the HEX channel was positive, the result was negative. When both channels were negative, the result was considered invalid, and the RT-RAA assay was redone. The time it took to analyze specific numbers of samples using a particular method was recorded.
Clinical samples were assessed for the presence of SARS-CoV-2 viral RNA using commercial COVID-19 quantitative RT-PCR kits (Easy Diagnosis Biomedicine Co, Wuhan, China, and DAAN GENE, Guangzhou, China in ZhouShan Customs, Easy Diagnosis Biomedicine Co, Wuhan, China and, BioGerm Medical Technology, Shanghai, China in Hangzhou Customs), according to the manufacturers’ instructions. RT-PCR was performed alongside the RT-RAA kits at the facility sites (Hangzhou and Zhoushan Customs), where the clinical samples were stored. All the PCR procedures were performed on an ABI 7500 Real-Time PCR Instrument (Applied Biosystems, Foster City, CA) provided by the local customs. The time required to analyze a specific number of samples using a given method was recorded.
The efficiencies of the COVID-19 nucleic acid detection kit (Fluorescence RT-RAA) and commercial RT-PCR nucleic acid detection kit were compared at the Hangzhou and Zhoushan customs. The parameters compared included the sensitivity, specificity, overall agreement rate, Kappa coefficient, and the time it took to complete 14, 28, 56, 98, and 196 samples using a specific method (12).
IBM SPSS Statistics (version 21, IBM Corporation, NY, USA) was used to perform all the statistical analysis. The results were analyzed using Kappa test and paired-samples t test, and a P-value less than 0.05 was considered statistically significant.
Analytical results of RAA were compared to those of RT-PCR for SARS-CoV-2 at Hangzhou Customs and Zhoushan Customs. Among the 506 samples collected by the Hangzhou customs and Zhoushan customs, 16 were positive, whereas 490 were negative as detected using the RT-RAA kit (Table 2). These results were consistent with the RT-PCR results. The sensitivity and specificity were 100%, the total coincidence rate was 100%, and the Kappa value was 1 (P<0.05) for both methods.
Results obtained using RT-RAA nucleic acid detection kits Results obtained using RT-PCR nucleic acid detection kits
P Positive Negative Total Positive 16 0 16 100 100 1 <0.05 Negative 0 490 490 Total 16 490 506 Note: Time comparison of simultaneous detection of different numbers of specimens by RT-RAA and RT-PCR at Hangzhou customs. Abbreviations: RT-RAA=real-time reverse transcription recombinase-aided amplification; RT-PCR=reverse transcriptase polymerase chain reaction.
Table 2. Results of SARS-CoV-2 analysis using RT-RAA versus RT-PCR detection kits (Hangzhou customs and Zhoushan customs).
A time comparison was performed for the simultaneous detection of different numbers of specimens using RT-RAA and RT-PCR at Hangzhou Customs. The detection time of the RT-RAA kit was significantly shorter than that of the RT-PCR kit for samples less than 200, especially in the detection of samples <100 (Table 3).
Sample size (a) 14 28 56 98 196 Detection time (RT-RAA), minutes 22 37 67 112 202 Detection time (RT-PCR), minutes 110 110 110 220 330 P <0.05 Abbreviations: RT-RAA=real-time reverse transcription recombinase-aided amplification; RT-PCR=reverse transcriptase polymerase chain reaction.
Table 3. Comparison of the detection time between RT-RAA and RT-PCR detection kits.
Nucleic Acid Extraction
Analysis of Clinical Samples Using Reference RT-PCR Methods at Customs
Comparing the Efficiency of RAA Versus RT-PCR for the Detection of SARS-CoV-2 RNA
Statistical Data Analysis