tetano
Editor, Senior Moderator
Anal Chim Acta
. 2022 Mar 22;1199:339585.
doi: 10.1016/j.aca.2022.339585. Epub 2022 Feb 7.
Importance of sample input volume for accurate SARS-CoV-2 qPCR testing
Yugan He[SUP] 1 [/SUP], Tie Xie[SUP] 1 [/SUP], Qihang Tu[SUP] 2 [/SUP], Yigang Tong[SUP] 3 [/SUP]
Affiliations
Abstract
Nucleic acid testing is the most widely used detection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Currently, a number of COVID-19 real-time quantitative reverse transcription PCR (qPCR) kits with high sensitivity and specificity are available for SARS-CoV-2 testing. However, these qPCR assays are not always reliable in detecting low viral load samples (Ct-value ≥ 35), resulting in inconclusive or false-negative results. Here, we used a Poisson distribution to illustrate the inconsistent performance of qPCR tests in detecting low viral load samples. From this, we concluded that the false-negative outcomes resulted from the random occurrences of sampling zero target molecules in a single test, and the probability to sample zero target molecules in one test decreased significantly with increasing purified RNA or initial sample input volume. At a given RNA concentration of 0.5 copy/μL, the probability of sampling zero RNA molecules decreased from 36.79% to close to 0.67% after increasing the RNA input volume from 2 to 10 μL. A SARS-CoV-2 qPCR assay with an LOD of 300 copies/mL was used to validate the improved consistency of the qPCR tests. We found that the false-negative qPCR results of clinical COVID-19 samples with a Ct ≥ 35 decreased by 50% after increasing the input of purified RNA from 2 to 10 μL. The consistency, accuracy, and robustness of nucleic acid testing for SARS-CoV-2 samples with low viral loads can be improved by increasing the sample input volume.
Keywords: Nucleic acid testing; Poisson distribution; SARS-CoV-2; Sample input.
. 2022 Mar 22;1199:339585.
doi: 10.1016/j.aca.2022.339585. Epub 2022 Feb 7.
Importance of sample input volume for accurate SARS-CoV-2 qPCR testing
Yugan He[SUP] 1 [/SUP], Tie Xie[SUP] 1 [/SUP], Qihang Tu[SUP] 2 [/SUP], Yigang Tong[SUP] 3 [/SUP]
Affiliations
- PMID: 35227385
- DOI: 10.1016/j.aca.2022.339585
Abstract
Nucleic acid testing is the most widely used detection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Currently, a number of COVID-19 real-time quantitative reverse transcription PCR (qPCR) kits with high sensitivity and specificity are available for SARS-CoV-2 testing. However, these qPCR assays are not always reliable in detecting low viral load samples (Ct-value ≥ 35), resulting in inconclusive or false-negative results. Here, we used a Poisson distribution to illustrate the inconsistent performance of qPCR tests in detecting low viral load samples. From this, we concluded that the false-negative outcomes resulted from the random occurrences of sampling zero target molecules in a single test, and the probability to sample zero target molecules in one test decreased significantly with increasing purified RNA or initial sample input volume. At a given RNA concentration of 0.5 copy/μL, the probability of sampling zero RNA molecules decreased from 36.79% to close to 0.67% after increasing the RNA input volume from 2 to 10 μL. A SARS-CoV-2 qPCR assay with an LOD of 300 copies/mL was used to validate the improved consistency of the qPCR tests. We found that the false-negative qPCR results of clinical COVID-19 samples with a Ct ≥ 35 decreased by 50% after increasing the input of purified RNA from 2 to 10 μL. The consistency, accuracy, and robustness of nucleic acid testing for SARS-CoV-2 samples with low viral loads can be improved by increasing the sample input volume.
Keywords: Nucleic acid testing; Poisson distribution; SARS-CoV-2; Sample input.