Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control

Abstract

Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan probe/primer sets for each one. A panel of 24 assays was prepared and detected via a microfluidic quantitative PCR (MFQPCR) system using integrated fluidic circuits (IFCs). The limit of detection of the panel was 6.25 copy/μL. Amplification-specificity was validated using several concentrations of reference materials and animal genomic DNA, leading to specific detection. In addition, target-specific detection was successfully achieved in a horse administered 20 mg of the EPO transgene via MFQPCR. Therefore, MFQPCR may be considered a suitable method for multiple-target detection in gene-doping control. To our knowledge, this is the first application of microfluidic qPCR (MFQPCR) for gene-doping control in horseracing.

Keywords: gene doping; horse; microfluidic qPCR; multiple-target detection; transgene.

Figure 1

Probe and primer design for microfluidic quantitative PCR (MFQPCR) detection. Two assays (SET1 and SET2) were designed for each transgene. Forward and reverse primers for MFQPCR were designed to target different exons. TaqMan probe for MFQPCR was designed to target exon/exon junctions. Forward and reverse primers for pre-amplification were designed to include the forward and reverse primers for MFQPCR. While forward and reverse primers for pre-amplification and MFQPCR may amplify PCR products, including genomic DNA regions, TaqMan probes do not anneal to the PCR product, including genomic DNA regions (A). TaqMan probes specifically anneal to PCR products having exon/exon junction sequences (B).

Figure 2

Relative concentrations of reference materials (RMs) among the NanoDrop One^C, Qubit dsDNA HS Assay and ddPCR using SET1 and SET2. Concentrations were measured twice at days 1 (A) and 2 (B). Values of NanoDrop, Qubit and droplet digital PCR (ddPCR) SET2 were calculated for a SET 1 ddPCR value of 1.

Figure 3

Cycle threshold (Ct) of each transgene on positive control sample (PCS)_10000 (12,500 copies), PCS_1000 (1250 copies), PCS_100 (125 copies), PCS_10 (12.5 copies), PCS_5.0 (6.25 copies) and PCS_2.5 (3.125 copies) based on SET1 (A) and SET2 (B) detections. The experiment was repeated 35 times, and the mean values are shown.

Figure 4

Image of microfluidic quantitative PCR (MFQPCR) detection. Positive control samples (PCS_10000, PCS_1000, PCS_100, PCS_10, PCS_5.0 and PCS_2.5); negative control samples (horse genomic DNA and Milli-Q) and plasma at: before administration, 15 min, 3 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 14 d, 21 d and 28 d after EPO transgene administering high, middle and low concentration of PCSs for each transgene, and spiked and recovered samples for each transgene were MFQPCR-amplified using SET1 (A) and SET2 (B) detections. Vertical axis is assays; CKM, EPO, FGF2, FST, GH1, IGF1, MSTN, PCK1, PDK4, PPARD, VEGF and ZFAT for SET1 (A) and SET2 (B) detections. Light yellow and orange indicate lower cycle thresholds (Cts) (high copy concentrations). Dark purple depicts high Cts (low copy concentrations). Black indicates nonamplification. NCS: negative control sample.