key: cord-0262097-fn0kgply authors: Guo, C.; Yao, L.; Chen, F.; Zhang, C.; Chen, W. title: Development and Performance verification of colloidal gold labeled SARS-CoV-2 antigen detection method for routine popular screening of COVID-19 with clinical samples in Poland and China date: 2021-11-24 journal: nan DOI: 10.1101/2021.11.22.21266719 sha: 40bf2b3c7650e00772b627ecc643969ef9f31beb doc_id: 262097 cord_uid: fn0kgply In this research, we have constructed and optimized the colloidal gold labeled lateral flow strip (LFS) for rapid detection of antigen of SARS-CoV-2 and rapid screening of COVID-19. Based on the constructed and optimized colloidal gold lateral flow strip, the parameters of the LFS have been well evaluated with the clinical samples in the professional labs. The screening performance have also been evaluated from the aspects including the CT values, age distribution and onset of symptoms. Finally, based on the detection results of 420 clinical samples, the LFS can achieve the screening of COVID-19 with the positive percentage agreement (PPA, sensitivity), negative percent agreement (NPA, specificity), the positive predictive value (PPV) and the negative predictive value (NPV) of 96.8%, 100%, 100% and 96.6%, respectively, indicating the powerful potential for practical screening applications in pandemic control. Of great significance, this developed SARS-CoV-2 antigen detection method has also been successfully utilized for screening of delta-variant of SARS-CoV-2. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.22.21266719 doi: medRxiv preprint 3 The first outbreak of COVID-19 in Wuhan, China, 2019 and the subsequent prevalence of COVID-19 pandemic have caused serious life and economic loss all over the world 1 . With the confirmed whole genomic sequence information, the current gold standard method for SARS-CoV-2 detection is still the real time reverse transcript quantitative PCR 2 . With the evolution of the virus and the effective control of the pandemic in most of the countries, real time PCR can meet the detection requirements of acute outbreak in specific cities. However, with the resumption of work and production in many countries, routine screening of COVID-19 has become an effective strategy to prevent the outbreak of the pandemic. The large-scale screening in school, hospital, airport, station, and prison has been extensively conducted in Europe and US et al. Under this circumstance, the nucleic acid-based detection methods absolutely cannot the meet the urgent and huge requirements of common populations. The easy operation procedures and simple results judgement of colloidal gold labeled lateral flow strips make it suitable for wide applications in high throughput screening of common populations [3] [4] [5] . Herein, we systematic optimized and screened lots of raw materials to construct the colloidal gold labeled COVID-19 antigen detection kit for rapid and easy detection of N protein of SARS-CoV-2. As we know that the coding gene of N protein is comparable conservative and the N . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.22.21266719 doi: medRxiv preprint 4 protein is comparable stable, which determine that the developed colloidal gold labeled COVID-19 antigen detection kit can also be adopted for rapid screening of delta variants. The antibody for gold nanoparticle labeling and antibody for coating on nitrocellulose membrane (NC membrane) as detect line were ordered from Chongqing Fantibdoy S&T Co. Ltd. The goat-anti-mouse was also ordered from the same provider. The NC membrane (ealon 120B) was purchased from the Ealon Membrane (Shantou, China) Co. Ltd. Other materials including the adhesive pad (300*60 mm), the conjugation pad (KB50) and the sample pad (8965) were bought from the Shanghai Jiening Biotechnol. Co. Ltd. Firstly, the colloidal gold nanoparticles were prepared according to our previous reports. Then K2CO3 was used to modify the pH of gold nanoparticles to conjugate with the antibody against N protein of SRAS-CoV-2. Then, the colloidal gold labeled antibody conjugates were further blocked with BSA (0.5%, wt.%) and reacted for 30 min. The mixture was centrifuged at 10000 r/min for 40 min and the supernatant was discarded. While the precipitates were redispersed in the suspension buffer (0.1M Tris+20% sucrose+5.6% trehalose+1.5% BSA， is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.22.21266719 doi: medRxiv preprint 5 pH=7.8), which was used for the preparation of conjugation pad at the spraying rate of 2 μL/cm and dried at 37 ℃ for 2 h. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.22.21266719 doi: medRxiv preprint 9 positive results. These results indicate that this developed method can be applied for COVID-19 screening in all populations with different ages. Besides, the detection results were further analyzed based on the classification of onset time of the symptoms. Detailed results expressed in Table 3 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.22.21266719 doi: medRxiv preprint developed SARS-CoV-2 Antigen detection kit can meet the requirement of practical routine screening of large populations. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Finally, the developed SARS-CoV-2 Antigen detection kit was also adopted for the screening of the delta variants of SARS-CoV-2. For the results shown in Figure 1 , the developed SARS-CoV-2 Antigen detection kit can realize the successful detection of clinical delta samples. Of note, . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.22.21266719 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. CRISPR-Cas12-based detection of SARS-CoV-2 It is made available under a perpetuity.is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.22.21266719 doi: medRxiv preprint