Method for quantifying the content of intestinal viruses in clinical samples

Kashnikov Alexander Yurievich

In the study of intestinal viruses, there is often a need for an absolute quantitative assessment of the viral load (VN). The determination of the number of virus units in a sample is possible only using methods based on the application of standards identical to the test sample. Transmission electron microscopy (TEM) is not the most accurate but effective method of quantitative analysis of HV. This method is based on the use of live viral culture (VVC) ‒ the most adequate of the existing international standards for the quantitative determination of VN. In recent years, the absolute version of real-time PCR (PCR-RV) has been preferred in the quantitative analysis of HV. However, this method is no less time-consuming than TEM and the relative version of real-time PCR. In this paper, we propose a simplified method for quantifying the content of a specific type of virus in a clinical sample. Its essence is to determine the units of viruses of this type present in the sample under study, according to a standard schedule constructed using samples taken as a standard, in relation to this type of virus. The method is based on the use of data obtained by a relative variant of PCR-RV, on the presence of a specific virus in the sample and its quantitative ratio to viruses of another type. This information can also be confirmed using the TEM technique. As a working method for obtaining quantitative data on HV in the methodology, using a standard graph, a simple-to-perform technique of NanoDrop spectrophotometry is proposed. This technique makes it possible to determine the HV in a pre-purified and concentrated virus-containing material by the concentration of nucleic acid (NC) isolated from the purified virus, which is quantitatively comparable to the virus content in the sample. The graphical method will allow to determine the HV in clinical samples with one or more types of viruses, separately for each type. The reliability of the information on the value of the HV calculated by the proposed method is confirmed by the quantitative data obtained using TEM. It will significantly simplify the quantitative analysis of HV in samples with the ability to determine the number of viral particles only on the basis of data on the concentration of viral genetic material obtained by NanoDrop spectrophotometry. Using the graphical method, it will be possible to determine the amount of virus in samples from 108 particles/ml and higher, and with a preliminary concentration-and with a lower concentration.

Keywords:transmission electron microscopy, viral nucleic acid, NanoDrop spectrophotometry, real-time PCR, threshold cycle, standard curve.