Automatic purification and concentration of biological samples for PCR analysis

Within the framework of the Marie Sklodowska-Curie Action- Individual Fellowship program, a project DENVPOC, led by Adriana Freire Lubambo (PhD), was developed at BforCure to create and design a semi-automatic method of sample pretreatment before PCR analysis.

PCR (polymerase chain reaction) is a method used to detect pathogens in different types of samples (blood, saliva, water…) via the identification of pathogen-specific nucleic acid sequences. It can be applied to different fields, like human and animal health, detection of microorganisms in the environment, defense and security. In this technique the quality of the starting sample is fundamental: it must not contain PCR inhibitors and the concentration of pathogens must be appropriate for detection. If the sample is not pure enough or the concentration is too low, the sample must be pre-treated to inactivate the inhibitors, concentrate the pathogens and lyse them in order to release the nucleic acids.

In this project, the research focused on RNA viruses such as SARS-CoV-2, as the years 2020-2021 are at the heart of the Covid19 global pandemic.
The objective was to develop a semi-automated protocol to pretreat a complex sample before performing PCR. To this end, an automated system was designed to concentrate and purify the sample, and the system was validated as a proof-of-concept for semi-automated pretreatment on SARS-CoV-2 RNA-enriched saliva.

Thanks to various iterations, chemical, mechanical and microfluidic tests, the pretreatment was performed in two steps:

1. First, a manual inactivation, through the use of a chemical lysis buffer,
2. and then, an automated and microfluidic purification and concentration with the pretreatment machine.

A decontamination protocol for the pretreatment machine was also established, in order to be able to process several samples in a row.

The 15-minute pretreatment protocol is intended to be integrated upstream of two products developed by BforCure, the Chronos Dx PCR platform and the Bfast detection kit. Chronos Dx is the real-time PCR instrument that enables ultra-fast nucleic acid detection by qPCR (less than 15 minutes*) thanks to its patented and proprietary Fastgene technology. The Bfast kits consist of PCR reagents developed by BforCure to perform analyses on Chronos Dx or other qPCR platforms.

Each element of this analysis chain is compact and mobile and thus allows to process samples rapidly and in a delocalized manner, close to the needs of the patients. Moreover, this can be extended to other types of samples and pathogens such as dengue detectable in blood, a matrix on which it is difficult to perform direct PCR.

In summary, this project was multidisciplinary and allowed to develop and bring into play different skills (chemistry, microbiology, microfluidics, electronics…) within an innovative start-up in full expansion.

*Data measured with Bfast [SARS-CoV-2]RT-PCR kit.

This project has been funded by the European Union’s Horizon 2020 Marie Sklodowska-Curie Action- Individual Fellowship program under the grant agreement n° 892834.