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

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.

Comparison between L and E gene amplification analytical methods for human papillomavirus typing.

OBJECTIVE: L and E6/E7 gene amplification analyses were compared to identify human papillomavirus (HPV) infection and verify the HPV type, with the intent to minimize HPV typing errors. METHODS: L1 gene verified HPV typing was accomplished via polymerase chain reaction (PCR) and membrane assays. Verification of HPV typing via E6/E7 genes was accomplished through nested multiplexed PCR. The results from 104 samples were compared. RESULTS: The rates of accordance and difference were 35% and 65%, respectively. For 29% of the analyses, nested multiplexed PCR was more diversified than the membrane assay. CONCLUSION: HPV can be classified into low-risk HPV and high-risk HPV groups. In parallel amplifications of the L and E genes is more efficient for accurate diagnosis in light of the different symptoms and attendant precautions of the risk groups.

Melanogenesis inhibitory effects of methanolic extracts of Umbilicaria esculenta and Usnea longissima.

The primary objective of this study was to assess the in vitro melanogenesis inhibitory effects of methanolic extracts of the edible and medicinal lichens, Umbilicaria (Gyrophora) esculenta and Usnea longissima. The quantities of the total phenolic compounds of methanolic extract of the two lichen extracts were determined to be 1.46% and 2.62%, respectively. In order to evaluate the antioxidative effects of the extracts, we also measured electron donating abilities (EDA) and lipid peroxidation rates. The EDA values measured by the reduction of 1.1''-diphenyl-2-picrylhydrazyl (DPPH) were 72.8% and 80.7% for the extracts, with SC50 (median scavenging concentration) values of 1.29+/-0.05 mg/ml and 1.03+/-0.06 mg/ml, respectively. The rates of inhibition of lipid peroxidation using linoleic acid were 92.1% and 97.3% for the extracts, with IC50 (median inhibitory concentration) values of 0.57+/-0.05 mg/ml and 0.53+/-0.06 mg/ml, respectively. The inhibitory rates of the extracts against tyrosinase were 67.4% and 84.8%, respectively. The extracts were shown to reduce melanin formation in human melanoma cells. Melanin contents in the samples treated with 0.01% and 0.1% U. esculenta were 47.1% and 31.2%, respectively, and those treated with 0.01% and 0.1% Usnea longissima were 51.1% and 34.9%, respectively, whereas a value of 54.0% was registered when ascorbic acid was utilized as a positive control. In addition to direct tyrosinase inhibition, it was determined that the lichen extracts affected the activity of tyrosinase via the inhibition of tyrosinase glycosylation. As a result, the methanolic extracts of U. esculenta and Usnea longissima evidenced melanogenesis inhibitory effects, which occurred via multiple routes.