Enhancing RT-PCR Throughput and Sensitivity through Large-Scale Sample Pooling Using a Nano-Hybrid Membrane.

Area of Science:

• Molecular Biology
• Virology
• Biomaterials Science

Background:

• Sample pooling enhances RT-PCR throughput for infectious disease surveillance during outbreaks like SARS-CoV-2 and influenza.
• However, pooling dilutes positive samples, reducing assay sensitivity and increasing false negatives, posing a challenge for accurate detection.

Purpose of the Study:

• To develop an innovative method that overcomes the sensitivity limitations of sample pooling in RT-PCR.
• To maintain high accuracy and reduce false negatives in large-scale viral detection using pooled samples.

Main Methods:

• A novel nano-hybrid membrane, SIMPLE (streamlined, simple, and inexpensive method for preconcentration, lysis, and nucleic acid extraction), was developed.
• This membrane integrates layered red blood cell membranes, polyethersulfone, and silica for sample enrichment.
• The method was tested with pooled COVID-19 samples of varying sizes.

Main Results:

• The SIMPLE method effectively prevented pooling-induced decreases in viral concentration.
• Cycle threshold (Ct) values in pooled samples remained comparable to those of individual positive samples.
• A Ct value reduction of approximately 2.6 was observed in pooled samples with a pool size of 6.

Conclusions:

• The SIMPLE nano-hybrid membrane technology offers a promising solution to enhance RT-PCR accuracy in pooled sample testing.
• This method significantly improves the efficiency and reliability of large-scale viral detection, particularly for SARS-CoV-2 and influenza surveillance.
• The approach addresses the critical trade-off between pool size and diagnostic sensitivity in molecular testing.