True or false: what are the factors that influence COVID-19 diagnosis by RT-qPCR?

Abstract

Introduction The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease has had a catastrophic impact on the world resulting in several deaths. Since World Health Organization declared the pandemic status of the disease, several molecular diagnostic kits have been developed to help the tracking of viruses spread. Areas Covered This review aims to describe and evaluate the currently reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) diagnosis kit. Several processes used in COVID-19 diagnostic procedures are detailed in further depth to demonstrate optimal practices. Therefore, we debate the main factors that influence the viral detection of SARS-COV-2 and how they can affect the diagnosis of patients. Expert Opinion Here is highlighted and discussed several factors that can interfere in the RT-PCR analysis, such as the viral load of the sample, collection site, collection methodology, sample storage, transport, primer, and probe mismatch/dimerization in different brand kits. This is a pioneer study to discuss the factor that could lead to the wrong interpretation of RT-qPCR diagnosis of SARS-CoV-2. This study aimed to help the readers to understand what very likely is behind a bad result of SARS-CoV-2 detection by RT-PCR and what could be done to reach a reliable diagnosis.

Diagnosis of Viral Infections: A Review of the Current Assays and the Prediction of the Future

“…Then, the master mix was split into 12 separate wells in a 96-well plate, and each of the four wells were mixed with 3 μL of the test sample, NTC, and PC each. After this process, the well plate was placed into a small box before moving to the room with the PCR machine to avoid any possible contamination or exposure to strong lights, as failures in sample collection and storing methodology could create false-positive or false-negative results (Lima, et al, 2022).…”

Diagnosis of Viral Infections: A Review of the Current Assays and the Prediction of the Future

“…Then, the master mix was split into 12 separate wells in a 96-well plate, and each of the four wells were mixed with 3 μL of the test sample, NTC, and PC each. After this process, the well plate was placed into a small box before moving to the room with the PCR machine to avoid any possible contamination or exposure to strong lights, as failures in sample collection and storing methodology could create false-positive or false-negative results [11].…”

A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase‐aided reverse transcription PCR

“…The internal reference target is an important indicator in the field of nucleic acid detection and is widely used in qPCR and some isothermal nucleic acid detection methods, which can monitor the whole process of sampling, nucleic acid extraction and amplification, effectively reducing false negative results and thus increasing the authenticity of the test results. [21][22][23] Multiplex RT-RAP showed high sensitivity and specificity, the detection sensitivity of multiplex RT-RAP for HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively, which were In conclusion, the proposed closed-tube multiplex RT-RAP is a rapid, highly specific, and highly sensitive nucleic acid detection platform, which has great potential to be used in the detection of various pathogens.…”