Sensitive detection of SARS-CoV-2

Rapid and sensitive detection of SARS-CoV-2 (proteins, RNA, DNA) and antibodies is crucially important for restricting the spread of disease, improving therapeutic outcome and controlling the return to activity. However, the number of virus particles in clinical samples may be very low, depending on type of sample and course of the infection, or in the case of in asymptomatic patients. Thus, a method to selectively concentrate the virus is required in order to enable accurate and reliable detection of minimal traces. This project will develop sensitive rapid tests (30-40 min) and procedures for concentrating and detecting SARS-CoV-2 and related molecules using modified magnetic beads (finally inert ones) conjugated to antibodies anti- SARS-CoV-2 virus, viral proteins or sequences of nucleic acids to concentrate the analyte in the detection area.

The discarding of infection with almost full accuracy is also critical to have a full control of this illness.

There are deep concerns laboratory tests providing incorrect diagnostics. Among other factors, the most common origin for these errors is an insufficient amount of viral material collected from the patient. Patient tested too early in the course of infection or improper sampling may result in a false negative. With the introduction of quick serology tests, the situation is even worse. Available quick tests have presented a sensitivity of 20-50%. This makes it difficult to control the virus' spread as either patient turned away from medical facilities or suspicious mild cases with incorrect diagnostics may become community vectors of infection. When faced with a highly infectious pathogen as SARS-CoV-2, even a small number of false negatives can have a potentially serious and widespread impact on a large population or new outbreaks after the situation is put under control, mainly if affecting a critical population line medical service. Therefore, accurate diagnosis is necessary so that hospitals and resources are allocated to real cases, detecting minimal traces of virus infections to be used even in the very early stages of the infection.

Viral capture will be performed according to protocols already designed and used successfully for the detection of Legionella or Hepatitis C virus. Briefly, the magnetic beads (volume of 50 μL containing 5 micrograms of nanoparticles) will be washed twice with phosphate-buffered saline (PBS). A nasal/nasopharyngeal aspirate specimen (preferably 0.5-1.0 ml) will be diluted with 500 μL of PBS and incubated with the washed magnetic beads for 20 minutes at room temperature. After applying a magnet, the supernatant will be discarded. The beads will be then washed three more times with PBS. Finally, the washed beads will be resuspended in PBS and analyzed by immunolabelling and viral RNA extraction followed by RT-qPCR. . Moreover, nucleic probes immobilized onto magnetic nanobeads may be directly used to detect the virus. An immunocapture method for clinical measurements of antibody levels in serum samples will also be developed as well as to detect traces of viral RNA in saliva. This will produce slower test but with an extremely high sensitivity to control the epidemic.

It could help identify recovered patients who could then donate their SARS-CoV-2 antibody-rich serum to help treat critically ill patients. Another key application would be to identify people who have developed likely immunity to the virus. They might be able to treat patients safely or take on other front-line jobs during the pandemic. Widespread sensitive antibody testing could also provide key data for efforts to model the course of the pandemic.

Such data could inform practical issues such as whether and how to reopen facilities that have been closed. Longer term antibody tests will also help researchers understand how long immunity to the virus lasts, a key issue for any future vaccine. Detection of a single molecule of ARN or ADN containing a sequence if the virus in nasal fluids may be too precise for diagnosis, but if it is present in blood, it may be established that there is some virus in the organism.

Several questions will come out when the gross of the pandemic has passed: how many COVID-19 cases have gone undetected? And are those who had mild cases of the disease immune to new infections? And are those who had mild cases of the disease immune to new infections? Could those cases slow the spread of the burgeoning pandemic? Answering those questions is crucial for understanding the pandemic spread and forecasting its future course. Our new approach may provide answers about the real number of COVID-19 infections and how deadly the virus has affected the population.