Mitra and DBS used in EPO transgene doping detection
by James Rudge, PhD, Technical Director, Neoteryx on Oct 25, 2021 9:00:00 AM
Using Dried Microsamples to Detect EPO Doping in Sports
An article published as a “short communication” in the February 2021 issue of the journal Drug Testing and Analysis by researchers at the French anti-doping agency, Agence Française de Lutte contre le Dopage (AFLD), demonstrated the utility of using dried blood microsamples to detect transgenic erythropoietin (EPO) doping in blood. The paper, titled "EPO transgene detection in dried blood spots for antidoping application" describes the evaluation of two TaqMan™ PCR assays specific for probing the exon 3-exon 4 junction from plasmid spiked blood. It was demonstrated that as little as 1000 transgene copies could be reliably amplified from 20 µL dried blood with >1 month room temperature stability.
The researchers conducted the study using two different approaches to dried blood sampling: dried blood spot (DBS) cards and Mitra® devices, which are based on volumetric absorptive microsampling. Mitra devices were demonstrated to have the edge in terms of sensitivity. The team, led by Alexandre Marchand, proposed a workflow where initial testing of each dried blood sample could be conducted using one of the TaqMan™ assays (custom) and any suspicious samples could be confirmed with the second (commercial).
Background on EPO Transgene Doping in Sports
What is EPO?
EPO is a naturally occurring peptide hormone in the body that (amongst other functions) stimulates erythropoiesis in bone marrow tissue. In doing so, EPO boosts aerobic function, which makes it attractive as a “performance enhancer” among competitive athletes. However, use of EPO as a performance enhancing substance is prohibited by the World Anti-Doping Agency (WADA) and is included on its List of Prohibited Substances. Nevertheless, EPO is reported to be the most used non-Specified Substance in the class of Peptide Hormones, Growth Factors, and Related Substances in sports. Indeed, doping with EPO grabbed global headlines in 2013 when it was discovered that disgraced champion cyclist, Lance Armstrong, had been secretly EPO doping during all seven of his Tour de France victories.
Why are researchers investigating methods of EPO detection?
To avoid detection of synthetic EPO, illegal doping laboratories have turned to using transgene vectors such as deactivated viruses or recombinant plasmids. Based on developments in gene therapy, human EPO DNA is inserted into the DNA of a vector, which is then introduced into the host (the doping athlete). In the case of EPO transgene doping, this is achieved by a simple infusion into the bloodstream to introduce the recombinant vector. As it enters the bloodstream, white blood cells take up the vector, exposing it to the cell’s protein synthesis machinery, resulting in transcription and translation of the EPO gene into host EPO protein.
Although the transgenic synthesis of EPO produces peptides are identical to naturally produced peptides, it is still possible to tell when someone has been genetically doped. The way to achieve this is to measure the sequence of Transgenomic DNA. This is because transgene’s base pair sequence is different to the host’s sequence for the gene of interest. The reason for this is that the sequence of many human genes, found in DNA, are not contiguous but are composed of both exons (coding regions) and introns (non-coding regions). However, when a gene is transcribed (during protein synthesis), introns are spliced out of the resulting pre-mRNA, thus forming the much smaller but now mature mRNA.
Due to vector size limitations, scientists use mature mRNA as a template to make Transgenomic copy DNA (cDNA). The exon-exon junctions, found only in the transgene, make ideal targets for quantitative polymerase chain reaction (qPCR) assays. It is this approach that Alexandre Marchand et al used to reliably measure 1000 copies of EPO cDNA (or less, in some cases). What is remarkable is that they achieved this result using extractions from only 20 µL of dried blood. They showed that this method could enable easier remote screening of athletes in future. In comparison, the paper commented that current WADA guidelines recommend collecting 1 mL of wet blood to detect 1500 copies (or less), which is more of a challenge for remote collection events.
EPO Doping Study Methods & Findings
Are dried blood spot techniques a viable EPO screening approach?The group evaluated two qPCR assays to establish a working range (25 to 1000 copies using mixtures of EPO transgene and human genomic DNA as well as negative controls (human DNA no transgene EPO). Both assays gave comparable results.
- To establish analytical specificity using the samples as above, they conducted melting curve analysis (MCA) as well as gel electrophoresis. The commercial TaqMan™ assay showed only one amplicon at the expected size, but the custom TaqMan™ produced, as well as the expected product, a smaller amplicon (low concentration) indicating a lower specificity.
- For the dried blood analysis, the group first made a serial dilution of plasmids, containing cDNA of transgenomic EPO (pEPO), ranging from 1 x 10 8 copies per µL down to 1 copy per µL, in EDTA blood.
- These blood samples were then spotted (20 µL) onto two different DBS papers (Nucleic Card™ and Whatman 903™) and also sampled onto Mitra™ (20uL). The three microsamplers were then extracted and analysed by the two qPCR assays as quadruplicates.
- To establish assay sensitivity and cut-offs, they also employed both positive and negative controls. Moreover, like the previous experiment to measure specificity, MCA and gels were also run for both TaqMan™ assays.
From the results obtained, the research group proposed the following workflow for dried microsamples:Use the custom assay as the initial pre-screen and when they have identified suspect samples, then use the commercial assay (due to its higher specificity) for a confirmation.
Conclusions of EPO Doping Study Authors
- Showed that EPO transgene could be detected in 20 µL of dried blood on Mitra devices and two DBS papers.
- Showed two TaqMan™ PCR assays work for detection of the transgene. One for initial screening and the other for confirmation.
- Able to measure down to 250 copies in blood dosed with a pEPO.
- In the future it could be possible to create a multiplex PCR using different fluorescence probes.
- Future advances in DNA sequencing may allow for probing of multiple exon-exon junctions and sell as genetic modifications.
This paper shows again how versatile dried matrix microsampling has become. Due to the continued advances in the molecular diagnostics field, scientists can aim to stay one step in front of sports dopers. Moreover, due to the sensitivity shown when using Mitra devices for these types of applications, it is conceivable to imagine that remote sampling can be used to monitor the efficacy and longevity of genetic therapeutics, too. It has already been demonstrated that Mitra devices with VAMS technology can be used to reliably measure small and large molecular weight drugs such as monoclonal antibody therapeutics, which opens the possibility of measuring multiple drug types and biomarkers from one Mitra cartridge.
This study paper was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director as curated content. For details about the study reviewed here, please refer to the original article published online at Drug Testing and Analysis.