Biotechnology

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https://hdl.handle.net/20.500.14540/123

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Browsing Biotechnology by Subject "Ct value"

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    Molecular Detection of Sars-Cov-2 RNA in Nasopharyngeal/Oropharyngeal Swab of Patient Without RNA Extraction
    (Department of Biotechnology, 2022) Karna, Suruchi
    RT-PCR is the gold standard method used till date for covid19 detection. Owing to the limited supply of SARS-CoV-2 RNA extraction kits in different health care facilities of Nepal, it results in enormous pressure to optimize reagent use, thereby affecting the overall diagnostic quality. This proposed research aimed to detect SARS-CoV-2 RNA from NPS through direct RT-qPCR technique omitting entire RNA extraction process. For this, 184 clinical NPS samples were obtained from Covid19 suspected patients who visited the Kirtipur Municipality-TU Biotech Corona Laboratory, and all subsequent steps were carried out there. These corresponding sample was subjected to RNA extraction followed by RT-qPCR as well as heat inactivated- RT-qPCR for validation. Eventually, their Ct values were compared wherein, the impact of heating temperatures and sample volume on assay sensitivity was also studied. The overall efficacy of these techniques was comparatively analyzed based on their Ct values. Heating NPS samples (n=184) for 20 min at 70 °C yielded a sensitivity, specificity, and accuracy of 93.3%, 96.7%, and 91.3% respectively. According to our paired T-test analysis, the mean Ct values of the N1 and RNase P genes were statistically significant at 95% CI (p<0.001), whereas the N2 genes were not (p>0.001). The results thus obtained was also compared with that of conventional RT-qPCR technique. Thus, a strong agreement using Cohen’s Kappa (κ=0.803) was found between two methods indicating reliability of heat inactivation assay. Therefore, direct RT-PCR might be a useful method for quickly identifying COVID19 suspects. This research offers a quick fix for the RNA extraction supply crunch. Furthermore, this emerging concept could even drastically lower costs and accelerate assay TAT by omitting the RNA extraction step.
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    S Gene target failure analysis in Covid positive patienus using Taqpath Covid-19 Assay
    (Institute of Science and Technology, Biotechnology, 2022) Acharya, Sushma
    The evolution of SARS-COV-2 resulted in the emergence of various variants of concern. A deletion at positions 69 and 70 of the spike glycoprotein precursor gene (S-gene), is associated with S gene Target Failure (SGTF) in a commonly used three gene diagnostic RTqPCR assay and is one of the mutations that characterize the omicron and alpha variants till now. The use of TaqPath COVID-19 CE-IVD RT-PCR kit with a non-detectable S gene target and Ct value≤37 for either the ORF1ab or N gene targets was classified as SGTF. In this study, nasopharyngeal swab samples of covid positive patients visiting TU Biotech Corona Laboratory were used. Of the total positive samples, 24.26% (304/1,253) of the samples with low cycle threshold (Ct) values were selected to access SGTF. The rate of SGTF detection was high in the end of January which slowly decreased towards the start of next month. Among the 304 samples, 16.11% (49/304) samples resulted in SGTF whereas 83.88% (255/304) samples showed non-SGTF. Highest number of SGTF was found in the sample having the Ct values in the range 30-35 for both N gene and ORF1ab. Therefore, Ct mean value of SGTF samples were found to be greater than that of non- SGTF samples. Among the SGTF samples, 69.38% (34/49) of them were male and 30.61% (15/49) were female. The mean age of SGTF individuals was 38.20 years ranging from 8 to 73 years old. In the study population highest number of SGTF cases 29/49 (59.18%) belonged to 25- 45 years age. Cough, fever and sore throat were the common symptoms in the SGTF patients. Among the SGTF samples the highest number of patients were vaccinated with Verocell followed by Covishield, Johnson and Pfizer vaccines. Detection of new variants of SARSCOV-2 with mutated spike protein through the SGTF approach helps in identifying circulating variants in the community. Keywords: SARS-CoV-2, RT-qPCR, SGTF, non-SGTF, Ct value