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Positive control synthesis method for COVID-19 diagnosis by one-step real-time RT-PCR.

ABSTRACT: BACKGROUNDS:The coronavirus disease 2019 (COVID-19) pandemic is still ongoing. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is regarded as a gold-standard method for the diagnosis of COVID-19. However, unexpected contamination of synthesized positive control samples included in COVID-19 test kits have increased the inconclusiveness of disease interpretation. Therefore, it is important to establish new methods for the preparation of reliable positive controls that are not affected by contamination for the accurate for diagnosis of COVID-19, but it still remains a challenge. METHODS:A new approach for producing synthetic positive controls using synthetic positive template (SPT) oligonucleotides was designed. SPT oligonucleotides contain probe binding and virus-irrelevant regions were used as templates for real-time PCR to evaluate the expression level of SARS-CoV-2 genes (RdRP, E, and N). The limit of detection (LOD) for individual SARS-CoV-2 genes by Ct values with different concentrations of SPT templates and genomic RNAs from SARS-CoV-2 infected samples was determined. RESULTS:LODs with SPT templates were >10^-15 (atto) M for RdRP, 10^-12 (femto) to 10^-13 (100 atto) M for E gene, and 10^-12 to 10^-14 (10 atto) M for N gene, respectively. Real-time RT-PCR assay using serially diluted genomic RNAs prepared from SARS-CoV-2 virus infected cultures showed that picogram quantities of RNAs is resulted in the LOD. The sensitivity of RdRP and E genes based on Ct values was less than that of N gene with this platform. CONCLUSION:This method significantly reduces the risk of false-positive reactions resulting from contamination in the synthesis procedures of positive control materials. Therefore, this approach could be integrated into the currrently available COVID-19 test kits and will provide a general method for preparing positive controls in the diagnosis of emerging RNA virus infections.

SUBMITTER: Cho H

PROVIDER: S-EPMC7550048 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Positive control synthesis method for COVID-19 diagnosis by one-step real-time RT-PCR.

Cho Hyejin H Jung Young Hwan YH Cho Hong Bum HB Kim Hee-Tae HT Kim Kwang-Sun KS

Clinica chimica acta; international journal of clinical chemistry 20201012

<h4>Backgrounds</h4>The coronavirus disease 2019 (COVID-19) pandemic is still ongoing. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is regarded as a gold-standard method for the diagnosis of COVID-19. However, unexpected contamination of synthesized positive control samples included in COVID-19 test kits have increased the inconclusiveness of disease interpretation. Therefore, it is important to establish new methods for the preparation of reliable positive contro ...[more]

PMID: 33058837

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Project description:BackgroundDengue is one of the leading causes of morbidity in tropical and subtropical regions and infection with any of the four dengue virus serotypes (DENV1-4) result in a wide range of clinical manifestations. Given the geographic expansion of DENV1-4, assays for serotyping are needed to be able to perform surveillance and epidemiological studies. In this study, we describe the design and validation of one-step real-time serotype-specific DENV RT-PCR assays.MethodsThe DENV1, DENV2, DENV3, and DENV4 RT-PCR assays were designed using all available whole genome DENV sequences in the NCBI nucleotide collection. Because of the high mutation rates of RNA viruses, the assays were performed in singleplex format to enable quick modifications to the primer and probe sequences when new genetic variants emerge. The analytical performance of the RT-PCR assays were evaluated using in vitro transcribed RNA and their specificity was determined by testing 24 DENV isolates, external DENV control panels and RNA preparation of non-DENV flaviviruses and non-dengue clinical samples. Additionally, the clinical performance of the serotype-specific DENV RT-PCR were compared to that of the CDC DENV-1-4 RT-PCR using 85 clinical samples collected from patients presenting with acute dengue.ResultsThe RT-PCR assays were found to be specific for their respective serotype and did not cross-react with other flaviviruses or human mRNA. All assays had a linear dynamic range of 10(2) to 10(6) copies/reaction with detection limits between 12 and 44 copies/reaction. When testing sera from 85 confirmed acute dengue cases, the serotype-specific DENV RT-PCR assays had 100 % positive agreement with the FDA-approved CDC DENV-1-4 RT-PCR assay performed in a singleplex format. Additionally 15 samples that tested negative in the CDC DENV-1-4- RT-PCR assay were found positive using the serotype-specific DENV RT-PCR assays.ConclusionsOur results suggest that these RT-PCR assays are useful alternatives to existing methods for serotyping DENV in clinical sera.

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Positive control synthesis method for COVID-19 diagnosis by one-step real-time RT-PCR.

Publications

Positive control synthesis method for COVID-19 diagnosis by one-step real-time RT-PCR.

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