Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger.

INTRODUCTION: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. METHODS: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. RESULTS: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. CONCLUSION: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.

Validación de una nueva estrategia para la identificación de variantes de SARS-CoV-2 mediante secuenciación del gen espiga por Sanger / Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger

Introducción: La aparición de múltiples variantes del SARS-CoV-2 durante la pandemia de COVID-19 es motivo de gran preocupación mundial. Hasta el momento, su análisis se ha centrado principalmente en la secuenciación de nueva generación. Sin embargo, esta técnica es costosa y requiere equipos sofisticados, largos tiempos de procesamiento y personal técnico altamente cualificado con experiencia en bioinformática. Para contribuir al análisis de variantes de interés y de preocupación, aumentar la capacidad diagnóstica y procesar muestras para realizar vigilancia genómica, proponemos una metodología rápida y fácil de aplicar, basada en la secuenciación Sanger de 3 fragmentos del gen que codifica para la proteína espiga. Métodos: Se secuenciaron 15 muestras positivas para SARS-CoV-2 con un valor de umbral de ciclo inferior a 25 por metodologías Sanger y secuenciación de nueva generación. Los datos obtenidos fueron analizados en las plataformas Nextstrain y PANGO Lineages. Resultados: Ambas metodologías permitieron identificar las variantes de interés reportadas por la OMS. Se identificaron 2 muestras como alfa, 3 gamma, una delta, tres mu, una ómicron y 5 cepas cercanas al aislado inicial del virus Wuhan-Hu-1. Según el análisis in silico, también se pueden detectar mutaciones clave para identificar y clasificar otras variantes no evaluadas en el estudio. Conclusión: Los diferentes linajes de interés y preocupación de SARS-CoV-2 se clasifican de forma rápida, ágil y fiable con la metodología de secuenciación de Sanger.(AU)

Introduction: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. Methods: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. Results: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. Conclusion: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.(AU)

[Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger]. / Validación de una nueva estrategia para la identificación de variantes de SARS-CoV-2 mediante secuenciación del gen espiga por Sanger.

Introduction: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. Methods: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. Results: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. Conclusion: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.

Validación de una técnica de PCR dúplex usando el gen E y RNasa P para el diagnóstico de SARS-CoV-2 / Validation of a duplex PCR technique using the gen E and RNase P for the diagnosis of SARS-CoV-2

Introducción: El estándar de diagnóstico para SARS-CoV-2 es la reacción en cadena de la polimerasa (PCR). La Organización Mundial de la Salud recomendó el protocolo de Charité-Berlín para el diagnóstico de COVID-19; esta metodología implica tres PCR, limitando la capacidad de procesamiento y retrasando los resultados. Con el fin de reducir estas limitaciones, se validó una PCR dúplex para la detección del gen E y RNasa P. Métodos: Se comparó el límite de detección, sensibilidad y especificidad de la técnica de PCR dúplex (gen E más RNasa P), comparada contra el estándar monoplex (gen E), en muestras de ARN de un aislado de SARS-CoV-2 y de 88 especímenes clínicos, con resultados previamente conocidos. Se determinó la repetibilidad y reproducibilidad de los valores de ciclos umbrales (cycle threshold [Ct]), en dos laboratorios independientes de la Facultad de Medicina de la Universidad de Antioquia, usando reactivos y equipos diferentes. Resultados: No hay diferencias significativas (p = 0,84) en los resultados de Ct entre ambas estrategias. Al utilizar como referencia el gen E amplificado en monoplex, el análisis de concordancia demostró fuerte similitud entre las dos estrategias, con un coeficiente kappa de Cohen de 0,89, una sensibilidad del 90%, y una especificidad del 87%. Conclusión: La PCR dúplex no afecta la sensibilidad y especificidad informadas por el protocolo Charité, Berlín, siendo una herramienta útil para el cribado de SARS-CoV-2 en muestras clínicas.(AU)

Introduction: Reverse transcriptase - polymerase chain reaction (RT-PCR) is the standard technique for SARS-CoV-2 diagnosis. The World Health Organization recommends the Charité-Berlin protocol for COVID-19 diagnosis, which requires triple PCR, limiting the process capability of laboratories and delaying the results. In order to reduce these limitations, a duplex PCR is validated for the detection of the E and RNase P genes. Methods: We compared the limit of detection, sensitivity and specificity of the duplex PCR technique (E gene and RNase P) against the monoplex standard (E gene) in RNA samples from a SARS-CoV-2 isolate and 88 clinical specimens with previously known results. The repeatability and reproducibility of the threshold cycle values (Ct) were determined in two independent laboratories of the Faculty of Medicine of the Universidad de Antioquia, using different reagents and real time instruments. Results: There were no significant differences in the Ct results between both techniques (p = 0.84). Using the monoplex PCR of E gene as a reference, the interrater reliability analysis showed similarity between the two techniques, with a kappa coefficient of 0.89, the sensitivity and the specificity of duplex PCR were 90% and 87%, respectively. Conclusions: Duplex PCR does not affect the sensitivity and specificity reported by the Charité, Berlin protocol, being a useful tool for SARS-CoV-2 screening in clinical samples.(AU)

Validation of a duplex PCR technique using the gen E and RNase P for the diagnosis of SARS-CoV-2.

INTRODUCTION: Reverse transcriptase - polymerase chain reaction (RT-PCR) is the standard technique for SARS-CoV-2 diagnosis. The World Health Organization recommends the Charité-Berlin protocol for COVID-19 diagnosis, which requires triple PCR, limiting the process capability of laboratories and delaying the results. In order to reduce these limitations, a duplex PCR is validated for the detection of the E and ribonuclease P genes. METHODS: We compared the limit of detection, sensitivity and specificity of the duplex PCR technique (E gene and Rnasa P) against the monoplex standard (E gene) in RNA samples from a SARS-CoV-2 isolate and 88 clinical specimens with previously known results. The repeatability and reproducibility of the threshold cycle values ​​(Ct) were determined in two independent laboratories of the Faculty of Medicine of the Universidad de Antioquia, using different reagents and real time instruments. RESULTS: There were no significant differences in the Ct results between both techniques (P = .84). Using the monoplex PCR of E gene as a reference, the interrater reliability analysis showed similarity between the two techniques, with a kappa coefficient of 0.89, the sensitivity and the specificity of duplex PCR were 90% and 87%, respectively. CONCLUSIONS: Duplex PCR does not affect the sensitivity and specificity reported by the Charité, Berlin protocol, being a useful tool for SARS-CoV-2 screening in clinical samples.

Validation of a duplex PCR technique using the gen E and RNase P for the diagnosis of SARS-CoV-2. / Validación de una técnica de PCR dúplex usando el gen E y RNasa P para el diagnóstico de SARS-CoV-2.

INTRODUCTION: Reverse transcriptase - polymerase chain reaction (RT-PCR) is the standard technique for SARS-CoV-2 diagnosis. The World Health Organization recommends the Charité-Berlin protocol for COVID-19 diagnosis, which requires triple PCR, limiting the process capability of laboratories and delaying the results. In order to reduce these limitations, a duplex PCR is validated for the detection of the E and RNase P genes. METHODS: We compared the limit of detection, sensitivity and specificity of the duplex PCR technique (E gene and RNase P) against the monoplex standard (E gene) in RNA samples from a SARS-CoV-2 isolate and 88 clinical specimens with previously known results. The repeatability and reproducibility of the threshold cycle values (Ct) were determined in two independent laboratories of the Faculty of Medicine of the Universidad de Antioquia, using different reagents and real time instruments. RESULTS: There were no significant differences in the Ct results between both techniques (p = 0.84). Using the monoplex PCR of E gene as a reference, the interrater reliability analysis showed similarity between the two techniques, with a kappa coefficient of 0.89, the sensitivity and the specificity of duplex PCR were 90% and 87%, respectively. CONCLUSIONS: Duplex PCR does not affect the sensitivity and specificity reported by the Charité, Berlin protocol, being a useful tool for SARS-CoV-2 screening in clinical samples.