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.
- Virome, microbiome, and metagenomics research using direct, non-culturable samples from humans, animals, and the environment
- Amplification to detect viruses and microbes in clinical specimens – especially when input nucleic acid is low or when rapid, random amplification is needed
- Agnostic amplification, or testing sample without prior sequence knowledge
- One-step whole genome or whole transcriptome amplification kits
- Amplification of clinical samples in the field, such as remote areas or during an outbreak
- Monitoring and public health surveillance research
- Rapid one-step or two-step protocol with few hands-on procedures with high sensitivity
- Can amplify either DNA or RNA from biological samples
- Offers nonbiased amplification of all nucleic acids in a sample
- Allows high-throughput processing of samples, including both culturable and non-culturable samples, and low to high yield nucleic acids
- Provides a broadly applicable method when using universal primers and eliminates the need for microorganism-specific primer design