Real-time RT-PCR in COVID-19 detection: issues affecting the results. (2020, August 21). PubMed Central (PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189409/

Michigan State University

Tufts University

COVID-19 detection via nucleic acid is perhaps one of the most rapid and stable technologies. A reverse transcriptase-coupled qPCR (RT-qPCR) is often considered as the rapid detection technique among with high sensitivity and specificity, particularly for identifying early infection. Using viral-specific forward and reverse primers of conserved viral sequence as a hallmark for detection, there were some cases of false negatives and false positives. This is mostly due to the fact that coronaviruses are known for rapid evolution and genetic drift, as mutations occurring in the primer target sequences were also frequent. Thus, mismatches between primers and target sequences often contribute to poorer accuracy. 
Other factors like sample sources and period of disease development also determine the accuracy of RT-qPCR readouts. Most common samples collected were from nasal or throat swabs, as they were the easiest to obtain without much patient discomfort. Another possible detection through collecting broncheoalveolar lavage fluid (BALF) could be more accurate, but required medical suction device and licensed operator. Other non-respiratory samples could be collected too, such as stool and blood. Regardless of which sample, all collected samples should be sent to testing site as soon as possible while being handled carefully to avoid false reads. Thus, sample timing and phases of infection also play significant roles in read outs. 


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