Dataset Information

Capillary electrophoresis of PCR fragments with 5´-labelled primers for testing the SARS-Cov-2.

ABSTRACT: Due to the huge demand for SARS-Cov-2 determination,alternatives to the standard qtPCRtestsare potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples.We isolated the naso-pharingealRNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5´-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks.The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. In conclusion, we describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5?h. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR and increase the testing capacity.

SUBMITTER: Gomez J

PROVIDER: S-EPMC7351060 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Capillary electrophoresis of PCR fragments with 5´-labelled primers for testing the SARS-Cov-2.

Gómez Juan J Melón Santiago S Boga José A JA Alvarez-Argüelles Marta E ME Rojo-Alba Susana S Leal-Negredo Alvaro A Castello-Abietar Cristian C Alvarez Victoria V Cuesta-Llavona Elías E Coto Eliecer E

Journal of virological methods 20200710

Due to the huge demand for SARS-Cov-2 determination,alternatives to the standard qtPCRtestsare potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples.We isolated the naso-pharingealRNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. O ...[more]

PMID: 32659241

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Project description:BackgroundHuman papillomavirus (HPV) E6/E7 type-specific oncogenes are required for cervical carcinogenesis. Current PCR protocols for genotyping high-risk HPV in cervical screening are not standardized and usually use consensus primers targeting HPV capsid genes, which are often deleted in neoplasia. PCR fragments are detected using specialized equipment and extra steps, including probe hybridization or primer extension. In published papers, analytical sensitivity is typically compared with a different protocol on the same sample set.A single-tube multiplex PCR containing type-specific primers was developed to target the E6/E7 genes of two low-risk and 19 high-risk genotypes (HPV6, 11 and 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82) and the resulting short fragments were directly genotyped by high-resolution fluorescence capillary electrophoresis.ResultsThe method was validated using long oligonucleotide templates, plasmid clones and 207 clinical samples of DNA from liquid-based cytology, fresh and formalin-fixed specimens and FTA Microcards® imprinted with cut tumor surfaces, swabbed cervical cancers or ejected aspirates from nodal metastases of head and neck carcinomas. Between one and five long oligonucleotide targets per sample were detected without false calls. Each of the 21 genotypes was detected in the clinical sample set with up to five types simultaneously detected in individual specimens. All 101 significant cervical neoplasias (CIN 2 and above), except one adenocarcinoma, contained E6/E7 genes. The resulting genotype distribution accorded with the national pattern with HPV16 and 18 accounting for 69% of tumors. Rare HPV types 70 and 73 were present as the sole genotype in one carcinoma each. One cervical SCC contained DNA from HPV6 and 11 only. Six of twelve oropharyngeal cancer metastases and three neck metastases of unknown origin bore E6/E7 DNA; all but one were HPV16. One neck aspirate contained atypical squames with HPV26.Analytical sensitivity in dilute plasmid mixes was variable.ConclusionsA primer-rich PCR readily detects the E6/E7 oncogenes of 21 HPV types in cellular and fixed tissue specimens. The method is straightforward, robust and reproducible and avoids post-PCR enzymatic and hybridization steps while detecting HPV with high clinical sensitivity in significant HPV-related neoplasia regardless of specimen type.

Dataset Information

Capillary electrophoresis of PCR fragments with 5´-labelled primers for testing the SARS-Cov-2.

Publications

Capillary electrophoresis of PCR fragments with 5´-labelled primers for testing the SARS-Cov-2.

Similar Datasets

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