![]() ![]() These standards could be optimized and set by government organizations, such as the Center for Disease Control and Prevention (CDC). In addition, since the Ct value is a non-standardized value and depends on the diagnostic reagent used, it may be necessary to standardize the Ct value according to the product for the same virus concentration. One way to judge ambiguous rRT-PCR results may be through detailed standard operating procedures performed by a centralized decision-making body consisting of specialists authorized by the government. Therefore, to overcome these limitations of the rRT-PCR method, the following strategies can be adopted. Furthermore, because the Ct value is inversely proportional to the amount of the target gene, there is also the disadvantage of a sample being interpreted as false negative in the early stages of COVID-19 infection without large amounts of virus multiplication, or depending on the accuracy of the swab. Therefore, when there is ambiguity in the Ct value, such as 34~36, the result may be interpreted as false negative or false positive depending on the Ct cut-off value. Although the Ct value in a rRT-PCR test is relatively accurate, error of 1~2 cycles are not uncommon in a Ct value depending on various factors, including the skill of the examiner. For example, although there are differences according to diagnostic reagents, a sample is usually judged positive for COVID-19 based on a Ct value of 35. However, the problem with a Ct-based diagnosis is that there is no absolute or constant Ct cut-off value, and Ct cut-off values are different for each diagnostic reagent even for the same gene. 5 In other words, the lower the Ct value of a specific gene, the more the gene exists in the sample. Ct is defined as the cycle number when the sample fluorescence exceeds a chosen threshold above the calculated background fluorescence. Secondly, the diagnosis of COVID-19 using rRT-PCR methods is not clearly classified as positive or negative, instead the diagnosis is made based on the threshold cycle (Ct) value. To overcome this drawback, it may be helpful to simultaneously use two or more rRT-PCR diagnostic kits that detect different viral genes. However, given that mutations occur frequently in SARS-CoV-2, the possibility of false negatives in the diagnosis of COVID-19 may be a disadvantage of rRT-PCR -based methods. However, interpreting the results may be challenging and requires attention.įirstly, because rRT-PCR methods usually detect only 2–3 of these genes, it has the advantage of rapid diagnosis. Since rRT-PCR-based assays usually detect only 2–3 of these genes, the assay allows for rapid testing and diagnosis. 4 Examples of RT-PCR diagnostic kits based on the aforementioned genes that are currently used in South Korea and the United States are listed in Supplementary 1 (Supplemental Digital Content 1, ). These countries have published their molecular diagnostic protocols and the primer/probe sequences on the World Health Organization website. Examples of a few countries and the target genes assayed are as follows: China (ORF1 ab, N), Germany (RdRP, E, N), Hong Kong (OLRF1b-nsp14, N), Japan (Pancoronavirus and multiple targets, S), Thailand (N), the United States (three targets in N), and France (two targets in RdRP). Most countries currently use rRT-PCR-based assays for the detection of COVID-19 infection. 3 Among them, the genes for the N and E proteins are used as the targets for amplification in the rRT-PCR assay combined with the open reading frame 1 (ORF1) ab, and the RNA-dependent RNA polymerase (RdRP) gene. ![]() The spike surface glycoprotein (S) mediates specific binding to the host cell receptors, the nucleocapsid (N) protein binds to the coronavirus RNA genome to make the nucleocapsid, the membrane (M) protein is the main structural protein that connects between the membrane and the capsid, and the small envelope (E) protein which is involved in the assembly and budding process of the coronavirus. The SARS-CoV-2 genome encodes four structural proteins. To understand the principle of the assay and the choice of primer sets used, some basic knowledge of COVID-19 biology is necessary. Currently, rRT-PCR is the most widely used diagnostic method for COVID-19. ![]()
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