Robustness of digital PCR and real-time PCR against inhibitors in transgene detection for gene doping control in equestrian sports.
Drug Test Anal
; 13(10): 1768-1775, 2021 Oct.
Article
in En
| MEDLINE
| ID: mdl-34270866
ABSTRACT
Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time PCR against gene doping. Digital PCR amplification was significantly inhibited by high concentrations of proteinase K (more than 0.1 µg/µl), ethylenediaminetetraacetic acid (more than 5 nmol/µl), and heparin (more than 0.05 unit/µl) but not by ethanol or genomic DNA. In addition, phenol affected droplet formation in the digital PCR amplification process. Real-time PCR amplification was inhibited by high concentrations of phenol (more than 1% v/v), proteinase K (more than 0.001 µg/µl), ethylenediaminetetraacetic acid (more than 1 nmol/µl), heparin (more than 0.005 unit/µl), and genomic DNA (more than 51.9 ng/µl) but not by ethanol. Although both PCR systems were inhibited by nearly the same substances, digital PCR was more robust than real-time PCR against the inhibitors. We believe that our findings are important for the development of better methods for transgene detection and prevention of false negative results in gene doping testing.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Polymerase Chain Reaction
/
Transgenes
/
Doping in Sports
Type of study:
Diagnostic_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
Drug Test Anal
Journal subject:
FARMACOLOGIA
Year:
2021
Document type:
Article
Affiliation country:
Japón