What are the primary PCR inhibitors in blood and fecal matrices?
The most common inhibitors in blood are heme, immunoglobulins (IgG), and anticoagulants like heparin. Fecal samples contain a more complex mixture, including bile salts, bilirubin, and complex polysaccharides from digested material, all of which can significantly suppress polymerase activity.
How does Franklin Biolabs validate its inhibitor removal process?
We employ a multi-step validation approach. The primary method involves spike-and-recovery experiments where a known quantity of a non-target internal amplification control (IAC) is added to each sample lysate before nucleic acid extraction. Consistent recovery of the IAC confirms the effective removal or neutralization of inhibitors on a per-sample basis.
Can your assays differentiate between residual input vector and replicated viral genomes?
Yes. For certain vectors, particularly RNA viruses or those with distinct replicative intermediates, we design strand-specific RT-qPCR assays. This allows for the specific quantification of newly synthesized genomic material, providing a direct measure of active replication versus simple persistence of the administered therapeutic.
What is the typical Lower Limit of Quantification (LLOQ) for viral shedding assays in these matrices?
The LLOQ is highly dependent on the vector, target sequence, and matrix composition. Our optimized protocols are designed to achieve high sensitivity appropriate for regulatory submissions, with assay-specific LLOQs established and justified during GxP validation.
Accurate quantification of viral shedding is a fundamental component of any IND-enabling safety package for viral-based therapeutics. However, complex biological matrices such as blood and feces are rich in potent PCR inhibitors that can lead to assay failure or under-quantification of vector DNA/RNA. This can compromise data integrity and misrepresent the biodistribution profile of a therapeutic candidate. Addressing these matrix effects requires specialized nucleic acid extraction protocols, inhibitor-resistant reagents, and rigorous, sample-specific internal controls to ensure data is reliable and defensible.
