Designing and making an in-house real-time RT qPCR kit for the detection of SARS-CoV- 2 infection at high sensitivity and low cost

Background The widespread use of accurate real-time RT-PCR tests for community individuals is a critical approach to managing the disease and reducing COVID-19 transmission effectively. In addition, serological ELISA assays are another essential tool for detecting and/or quantifying SARS-CoV-2 IgG and IgM antibodies or for screening for SARS-CoV-2 infection. Objectives The objective of the current study is to develop an in-house multiplex real-time RT-qPCR kit for molecular detection of SARS-CoV-2 N and E genes . Materials and methods A total of 100 samples from the Central Public Health Laboratory were analyzed: 50 tested positive for SARS-CoV-2 RNA and 50 tested negative. Results The in-house multiplex qPCR assay showed a significant association at P value 0.01 between the in-house N gene CT Value and the commercial N gene CT value, but there was no significant association at P value 0.09 between the in-house E gene CT Value and the commercial E gene CT Value. The in-house-designed RT-qPCR-E gene had a sensitivity of 96% and a specificity of 98.11%. The in-house-designed RT-qPCR N gene had a sensitivity of 92.31% and a specificity of 98.11%. Overall, or combined, sensitivity and specificity of the in-house-designed RT qPCR assay were 96.08% and 100.00%, respectively. Conclusions The in-house-designed multiplex real-time RT-qPCR kit was more sensitive than the commercial kit for detecting SARS-CoV-2, and the in-house ELISA kit showed acceptable sensitivity and specificity, given its very low cost. The in-house designed kits were far less expensive than the commercial kits. This study demonstrated a broad range of viral loads, which means some infected individuals with low viral loads might be below the limit of detection of commercial assays.