Validación y evaluación de una prueba de RT-PCR en tiempo real in house para la detección de SARS-CoV-2 usando un gen específico RdRp y control endógeno GAPDH

Abstract

Se validó y evaluó un método de RT-PCR en tiempo real usando cebadores y sondas específicas para los genes RdRP de SARS-CoV-2 y GAPDH de humanos; este último fue usado como control endógeno. Se evaluó la especificidad y sensibilidad; además, se evaluó otros parámetros como la robustez, la repetibilidad, reproducibilidad, comparabilidad y el límite de detección. La sensibilidad, especificidad, los valores predictivos positivo y negativo, la robustez, comparabilidad y la repetibilidad-reproducibilidad de la prueba de RT-PCR en tiempo real dúplex fue de 100%, con un límite de detección de 100 copias/μL, de acuerdo con los criterios de aceptación establecidos para validación del protocolo. Esta prueba estandarizada es una buena alternativa para el diagnóstico de COVID-19; además, la prueba fue aplicada de manera exitosa en personas sospechosas de la enfermedad permitiendo controlar el número de falsos negativos.

Development and Optimization of a Multiplex Real-Time RT-PCR to Detect SARS-CoV-2 in Human Samples

“…However, during the first year of the pandemic, many Latin American university research laboratories transferred their molecular skills to SARS-CoV-2 diagnosis, helping public health authorities to follow and control the virus transmission. For example, Peruvian academic and researchers standardized and validated a multiplex qPCR [ 16 ], while Ecuadorian and Uruguayan universities developed new qPCR systems that reached optimal clinical performance [ 17 ]. In Colombia and Brazil, university laboratories improved alternative low-cost multiplex PCR or Sybr green protocols to confirm positive samples [ 11 , 18 , 19 ].…”

What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes

“…The GAPDH gene produces a constitutive protein involved in many basic cellular functions such as glycolysis, DNA replication, nuclear RNA export, repair and apoptosis [14]. GAPDH is one of the best reference genes, but it is not ideal, as the expression of this gene is not identical in every tissue.…”

“…For throat swabs, however, it does not seem to matter, especially since we do not expect a quantitative result but a qualitative one, which indicates the presence of human RNA in the analyzed material. Other authors, for example, Rojas-Serrano or Wong et al, also believe that the gene serves as a good internal endogenous control to identify the possibility of obtaining false-negative results in the diagnosis of SARS-CoV-2 [14].…”

Diagnostic performance of a point-of-care molecular system for the detection of SARS-CoV-2 in Peru

“…RNA extraction was performed according to the manufacturer’s specifications with the QIAamp Viral RNA Mini kit either manually or using the QIACUBE® automated kit. For SARS-CoV-2 detection, the extract was subjected to real-time RT PCR-Charité protocol 3 and real-time RT-PCR using primers and probes specific for SARS-CoV-2 RdRP and human GAPDH genes 12 . Multiplex real-time RT-PCR in TaqMan system (qRT-PCR_Multiplex) was used for the detection of influenza A and influenza B viruses ( 13 .…”