Widespread testing is required to limit the current public health crisis caused by the COVID-19 pandemic. Multiple tests protocols have been authorized by the food and drugs administration (FDA) under an emergency use authorization (EUA). The majority of these protocols are based on the gold-standard RT-qPCR test pioneered by the U.S. Centers for Disease Control and Prevention (CDC). However, there is still a widespread lack of testing in the US and many of the clinical diagnostics protocols require extensive human labor and materials that could face supply shortages and present biosafety concerns. Given the need to develop alternative reagents and approaches to provide nucleic-acid testing in the face of heightened demand and potential shortages, we have developed a simplified SARS-CoV-2 testing protocol adapted for its use in research laboratories with minimal molecular biology equipment and expertise. The protocol utilizes TRIzol to purify the viral RNA from different types of clinical specimens, requires minimal BSL-1 precautions and, given its high sensitivity, can be easily adapted to pooling samples strategies.

Widespread testing is required to limit the current public health crisis caused by the COVID-19 pandemic. Multiple tests protocols have been authorized by the food and drugs administration under an emergency use authorization (EUA). The majority of these protocols are based on the gold-standard RT-qPCR test pioneered by the U.S. Centers for Disease Control and Prevention. However, there is still a widespread lack of testing in the US and many of the clinical diagnostics protocols require extensive human labor and materials, such as RNA extraction kits, that could face supply shortages and present biosafety concerns. Given the need to develop alternative reagents and approaches to allow nucleic-acid testing in the face of heightened demand and potential shortages, we have developed a simplified SARS-CoV-2 testing protocol adapted for its use in laboratory research with minimal molecular biology equipment and expertise. The protocol requires minimal BSL1 biosafety level precautions and facilities.

The HIV-1 genome is initially transcribed as a single primary RNA that undergoes extensive splicing to produce over 40 linear and 15 circular RNA (circRNA) isoforms sharing common sequences. Conventional methods for circRNA detection, such as Northern blotting and hybridization-based assays, are inadequate for distinguishing specific circRNA isoforms when multiple circular and linear species originate from the same transcript. We previously identified 15 HIV-1 circRNAs generated by backsplicing and demonstrated that some enhance viral replication by sequestering cellular miRNAs. PCR-based approaches using divergent primers (RT-qPCR) offer greater specificity for detecting individual circular RNAs under these conditions. Building on this, we have developed a TaqMan qPCR assay capable of specifically detecting 14 HIV circRNA isoforms using backsplicing junction-directed divergent primers coupled to a hydrolysis probe for signal confirmation. Compared with matched SYBR Green assays, the TaqMan platform showed lower background in non-infected controls and reduced variance across donor-derived samples. This method provides a robust platform for selective and qualitative analysis of HIV-1 circRNAs.