Loop‐mediated isothermal amplification (LAMP) as an alternative to PCR: A rapid on‐site detection of gene doping

Abstract

Innovation in medical research has been diverted at multiple occasions to enhance human performance. The predicted great progress in gene therapy has raised some concerns regarding its misuse in the world of sports (gene doping) for several years now. Even though there is no evidence that gene doping has ever been used in sports, the continuous improvement of gene therapy techniques increases the likelihood of abuse. Therefore, since 2004, efforts have been invested by the anti-doping community and WADA for the development of detection methods. Several nested PCR and qPCR-based strategies exploiting the absence of introns in the transgenic DNA have been proposed for the long-term detection of transgene in blood. Despite their great sensitivity, those protocols are hampered by limitations of the techniques that can be cumbersome and costly. The purpose of this perspective is to describe a new approach based on loop-mediated isothermal amplification (LAMP) for the detection of gene doping. This protocol enables a rapid and simple method to amplify nucleic acids with a high sensitivity and specificity and with a simple visual detection of the results. LAMP is already being used in clinical application for the detection of viruses or mutations. Therefore, this technique has the potential to be further developed for the detection of foreign genetic material in elite athletes.

DNA Microsystems for Biodiagnosis

“…Since this analytical method is still mainly used in research laboratories, new approaches are being investigated to make it practical, such as developing a portable detection prototype based on Arduino, an open electronics micro controller board [81]. Currently, LAMP is more accurate than quantitative-PCR for the detection of meningococcal infections in children [82], gene doping therapies [83], peanut allergens in processed food [84], acute viral necrobiotic infections in scallops [85], and Staphylococcus pseudintermedius bacterial infections in canines [86].…”

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…A potential alternative to these was suggested by Salamin et al, who discussed the utility of a loop‐mediated isothermal amplification (LAMP) approach for routine doping controls. Being a rather recent complement to clinical analyses, no proof‐of‐concept data concerning its application in sports drug testing exists; however, as the underlying strategy appears compatible with existing approaches (utilizing primers that target synthetic exon/exon junctions of EPO cDNA for example), an additional tool might become available to support anti‐doping methods concerning future gene doping issues …”

“…Extensive debates have continued around the topic of genetic predisposition as well as gene manipulation in sport, and rather different issues have been discussed with one calling into question the rules for who may compete in women's sports, 131 another addressing the FIGURE 2 Info box on particularly relevant observations relevance of genes and an athlete's environment in an individual's development from a talented athlete to a champion, 132 and the necessity and means to monitor gene doping practices in elite sport. 133 A variety of test methods targeting transgenic DNA in doping control samples have been established, largely relying on conventional PCRbased strategies. A potential alternative to these was suggested by Salamin et al, who discussed the utility of a loop-mediated isothermal amplification (LAMP) approach for routine doping controls.…”

A next-generation sequencing method for gene doping detection that distinguishes low levels of plasmid DNA against a background of genomic DNA

“…Although one can only speculate about the manner of administration, the most likely method would be injection of transgenes into the skeletal muscle in the form of viral constructs, after which the biochemical machinery of the cell would be recruited to express the transgene [3][4][5]. The most reliable assay to detect this form of gene doping would require a muscle biopsy, but such an invasive procedure is not appropriate [4,5]. However, in this scenario, small amounts of transgenes will leak into the bloodstream, and these can be isolated from a huge excess of genomic DNA (gDNA).…”

“…Currently published methods for detection of gene doping use PCR-based methods or loop-mediated isothermal amplification (LAMP) that target unique sequences in a doping gene corresponding to exon-exon junctions in the intron-less transgene [3,[5][6][7][8][9][10][11][12][13]. However, because the exon-exon junctions of doping genes are known and the short PCR primers are even interrupted by the slightest change of the sequence, it is relatively simple to evade detection using current PCR-based methods by modifying the doping gene with for example synonymous mutations, which will then give a false-negative result.…”