New molecular methodology for sensitive detection and quantification of mutant strains / variants of SARS-CoV-2 in wastewater samples using a series of molecular methods and next generation sequencing (Next Generation Sequencing / NGS) has been developed at the National and Kapodistrian University , through the collaboration of Research Units of the Departments of Biology, Medicine and Chemistry, in the context of research with scientific supervisor Professor Andreas Skorilas
This hypersensitive methodology is particularly important because it shows the presence and percentage of mutants in the total population of a city.
In addition, it detects in time, within 2 days, new mutations and strains / variants that have just begun to enter the population. This makes genomic surveillance of new strains / variants of SARS-CoV-2, as well as other viruses or bacteria, easier and more reliable, providing the necessary information for epidemiological surveillance as well as the redesign of new vaccines and drug protocols. .
The methodology meets the requirements of the relevant recommendation of 15-3-2021 of the European Commission for the common approach in establishing systematic monitoring of SARS-CoV-2 and its variants in wastewater. In addition, it detects new strains / variants in time that begin to enter the community.
The relevant article entitled “Novel Nested-Seq Approach for SARS-CoV-2 Real-time Epidemiology and In-depth Mutational Profiling in Wastewater” was published in the prestigious international scientific journal “International Journal of Molecular Sciences”, and has been submitted by EKPA Application for National and International Patents.
With the application of the new methodology by an interdisciplinary team of EKPA consisting of Andreas Skorilas, Nikolaos Thomaidis, Margariti Augeri, Athena Markou, Evi Lianidou, Dimitrios Gourgiotis, Theodora Psaltopoulou, Panagiotis Adamoparos, Lamparos Galoparos Maamaparos Galomoros Maamaparos Galoporos Ma , Katerina Kontou, Vassilis Kape, Nikolaos Voulgaris and Thanos Dimopoulos (Rector of EKPA), the analysis of mutant strains of SARS-CoV-2 in wastewater samples of the Attica Regional Unit has been completed, collected on a daily basis during the period 01-26 / / 2021 from the Wastewater Treatment Center of Psyttalia (KELD). The specific samples were tested for specific mutations – genetic markers (genetic markers) of the entire S gene (Spike) (> 50 genetic markers) for the variants of special interest (Variants of Concern – VOC) and the variants under investigation (Variants of Interest – VOI).
Foundings:
a. Samples for the period 1-15 / 12/2021
The results of the analysis showed the predominance of the Delta strain (B.1.617.2 lineage) at a rate of> 99%.
More specifically, the presence of specific mutations (genetic markers) was detected:
Delta strain (B.1.617.2 lineage) at> 99% of the total sequences.
– No specific genetic markers of the Omicron strain were detected (B.1.1.529 lineage)
– No specific mutations of VOCs: Beta (B.1.351) and Gamma (P.1), as well as VOIs: Mu (B.1.621) and Lambda (C.37) were not detected
b. Samples for the period 16-26 / 12/2021
The results of the analysis showed the significant increase in the percentage of genetic markers of the Omicron strain (B.1.1.529 lineage) by 34.4%, and the simultaneous significant decrease of the corresponding genetic markers of the Delta strain (B.1.617.2 lineage).
More specifically, it was found:
– the presence of specific markers (genetic markers) of the “receptor-binding domain / RBD” of Spike of the Omicron strain (B.1.1.529 lineage) at a rate of 34.4% (genetic markers: G339D, S371L, S373P, K417N, N440K, G446S)
– the presence of specific markers (genetic markers) outside the RBD part of Spike of the Omicron strain (B.1.1.529 lineage) at a rate of 7.9%
(genetic markers: P681H, N856K, N969K, L981F)
– No specific mutations of VOCs: Beta (B.1.351) and Gamma (P.1) as well as VOIs: Mu (B.1.621) and Lambda (C.37) were detected
In addition, to assess the daily change of some of the mutant strains found and confirmed by the next generation (NGS) sequencing of genetic mutants in the effluent, analysis was performed with five different RT-PCR methodologies. The specific methodologies allow the detection of mutations: a) N501Y (strains A, B, C, O), b) delH69 / V70 (strains A, O), c) K417N (strains B, O), d) K417T (strain C), and e) P681R (strains D, K). Based on the specific results of the analyzes, it appears that the Omicron strain / variant is detected in the samples from 20 to 23 December, while in the samples from 24 to 28 December, a significant increase is observed.
The overall findings of the gene analysis show that the mutant strain Omicron shows a strong potential for its dominance in the community with the gradual replacement of the prevailing Delta strain to date. At the same time, the sequence of the virus genome highlights significant variability in the percentage of specific mutations of the S gene encoding the Spike protein, emphasizing their different importance and significance in the behavior of the virus, as well as in its reliable and specific determination.
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