Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Abstract

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

Comparison of Manual and Automated Nucleic Acid (RNA) Extraction Methods for the Detection of SARS-CoV-2 by qRT-PCR

“…A similar study observed no significant difference in the RT-PCR positivity rate between the two methods statistically [ 18 ]. Another study done in Brazil observed 100% sensitivity in automated extraction when compared to manual and rapid extraction methods [ 13 ].…”

Evaluation of the time, labor, and money required for manual and automated nucleic acid (RNA) isolation for the detection of SARS-COV-2 by QRT-PCR using the qiamp viral RNA mini kit and kingfisher flex

“…It's interesting to note the varying findings across different studies. One study observed no significant difference in the RT-PCR positivity rate between two different methods, 6 suggesting comparable diagnostic performance. In contrast, a study in Brazil reported 100% sensitivity in automated extraction when compared to manual and rapid extraction methods, 11 indicating the reliability of automation in their context.…”

“…Automation outperforms manual extraction for highthroughput RNA isolation. [4][5][6] In a pandemic, molecular laboratories without automated nucleic acid extraction devices need trustworthy viral RNA extraction procedures. SARS-CoV-2 RNA recovery from nasopharyngeal swabs will be evaluated and optimized using two commercial kits: one with manual extraction and the other with automation.…”