One-Step Random Amplification Method to Detect Extremely Low Input Nucleic Acids for Virome, Microbiome, and Metagenomics in Clinical and Biological Specimens

Clinical and biological specimens often contain microbial nucleic acid in extremely low quantities, presenting a significant challenge for the detection of viral and bacterial pathogens. This also prevents direct sequencing of non-culturable samples using next-generation sequencing (NGS). Currently, NGS library preparation on most platforms requires 0.1 ng to 10 µg of DNA or cDNA, while microbial or viral nucleic acids in clinically relevant specimens, such as blood, serum, respiratory secretions, cerebral spinal fluid, and stool, often contain less than 0.1 ng.

CDC developed a rapid random amplification method capable of amplifying DNA and/or RNA inputs of less than 0.001 ng from biological samples. CDC’s method produces amplicons in amounts sufficient for NGS platforms. It allows non-biased universal amplification of all nucleic acids in a sample. Compared to traditional specific PCR (polymerase chain reaction), this technology amplifies unknown microorganisms without prior sequence knowledge and eliminates the need for microorganism-specific primer design. The method is rapid and requires minimal hands-on effort, while offering high sensitivity, time-savings, and high-throughput processing for random nucleic acid amplification.

Pilot experiments showed that low-yield clinical specimen viral RNA not amplified by other commercial random-amplification kits was amplified by this method and successfully sequenced. Thus far, CDC has achieved whole genome sequencing of a variety of viruses directly from clinical specimens, including but not limited to enterovirus (EV-D68, CV-A24, and EV-A71), cosavirus, parechovirus, and norovirus. Additional virus studies are underway.