Highly sensitive detection of SARS-CoV-2 RNA by multiplex rRT-PCR for molecular diagnosis of COVID-19 by clinical laboratories

Abstract

Background: The detection of SARS-CoV-2 RNA by real-time reverse transcription-polymerase chain reaction (rRT-PCR) is used to confirm the clinical diagnosis of COVID-19 by molecular diagnostic laboratories. We developed a multiplex rRT-PCR methodology for the detection of SARS-CoV-2 RNA. Methods: Three genes were used for multiplex rRT-PCR: the Sarbecovirus specific E gene, the SARS-CoV-2 specific N gene, and the human ABL1 gene as an internal control. Results: Good correlation of C q values was observed between the simplex and multiplex rRT-PCR methodologies. Low copies (< 25 copies/reaction) of SARS-CoV-2 RNA were detected by the novel multiplex rRT-PCR method. Conclusion:The proposed multiplex rRT-PCR methodology will enable highly sensitive detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical laboratory technicians.

Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay

“…ND, not detected. less sensitive when using the RNA positive control (Shirato et al, 2020;Ishige et al, 2020). In this study, we also confirmed that the N1 site of the NIID assay has a limit of detection of 10 copies of the DNA positive control.…”

A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2 Infection: High Sensitivity and Increased Testing Capacity

“…One-step qRT-PCR diagnostic sensitivity is influenced by multiple factors, such as the selection of the target genes, the design of the primer and probe sets and their concentration, the efficiency of the enzymes used, and the method of extraction and storage media [ 5 ]. Therefore, validation experiments are necessary to optimize this method for detection of viral RNA in human specimens [ 6 ]. The reported multiplex qRT-PCR method is based on the combined multitarget amplification of two fragments ( N1 and N2 ) of the SARS-CoV-2-specific N gene.…”

Microbiological Diagnosis of Covid-19 Disease in Children

“…Ayrıca daha bol eksprese edilen ACTB ve GAPDH genleri de internal kontrol için kullanılabilmektedir. Fakat PCR'da, oldukça bol hedeflerin amplifikasyonu, daha az olan hedeflerin amplifikasyonunu önleyebilir ve olasılıkla hedef saptama duyarlılığını azaltabilir (12) . etkilenir ve klinik ortamlarda bu metodoloji kullanılmadan önce optimizasyon deneyleri gereklidir (12) .…”

“…Fakat PCR'da, oldukça bol hedeflerin amplifikasyonu, daha az olan hedeflerin amplifikasyonunu önleyebilir ve olasılıkla hedef saptama duyarlılığını azaltabilir (12) . etkilenir ve klinik ortamlarda bu metodoloji kullanılmadan önce optimizasyon deneyleri gereklidir (12) . Bunun yanında virüsün mutasyona uğraması ve PCR inhibisyonu gibi sorunlar da yanlış negatifliği doğurabilmektedir (3) .…”