Validation Of Multiple Reference Genes As The Internal Control For Direct One-Step RT-Qpcr From Saliva Samples

Abstract

Saliva is one of the prospective samples used to identify respiratory infections and diagnose human diseases, including diabetes, cardiovascular disease, and cancer. Saliva has a significant benefit over various other samples, such as serum and nasopharyngeal swab (NPS), because it is collected quickly and painlessly. In addition, we require a rapid and accurate diagnostic test for viral detection to respond and prevent infectious diseases immediately before spreading. Therefore, our lab develops a direct one-step RT-qPCR from saliva samples, which is a rapid and cost-effective test that might be utilized in the case of a future pandemic. Nevertheless, the significant factor in RT-qPCR is the internal control gene, which prevents false negative results and ensures a reliable assay result. Typically, housekeeping genes (HKG) were used as ICGs because their expression was assumed to be stable across various tissue types, ages, and therapies. However, housekeeping genes’ expression stability varies depending on cell type, biological conditions, and experimental conditions. Researchers did not validate housekeeping genes as the internal control for direct RT-qPCR from saliva samples; consequently, it is critical to verify a stable gene as the internal control gene for direct one-step RT-qPCR. In this study, we discovered that RNase P is the stable gene that might serve as our internal control for the assay. Unfortunately, the RNase P primer from the CDC can bind to DNA; therefore, it is inappropriate to use it in direct one-step RT-qPCR from whole saliva. Furthermore, our group designs and proves novel primers for the RNase P gene, which targets RNA exclusively. Future development of the direct one-step RT-qPCR assay for infectious disease detection will be facilitated by the evaluation and validation of the internal control gene RNase P.