Sex determination in the wild: a field application of loop‐mediated isothermal amplification successfully determines sex across three raptor species

Huang

et al. 2017

Sci Rep

102

This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn2+ dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25–45 bp, beacon concentration of 0.6–1 pmol/μL, and reaction temperature of 60–65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product.

mentioning

confidence: 99%

Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

Huang

et al. 2017

Sci Rep

102

Drug Testing and Analysis

“…[42] In a different context, Centeno-Cuadros et al developed a LAMP protocol for sex determination of raptor species. [43] In comparison with PCR-based methods, LAMP amplification demonstrates better sensitivity and specificity. Wang et al demonstrated that this method was more sensitive and reliable than conventional PCR or multiplex for the detection of Arcobacter species.…”

Section: Loop-mediated Isothermal Amplification (Lamp)mentioning

confidence: 99%

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

Salamin

Kuuranne

Saugy

et al. 2017

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.

“…Moreover, product detection can be achieved directly within the reaction tube by a diagnostic color change and fluorescence, eliminating the need for electrophoretic techniques and related equipment (Centeno-Cuadros, Abbasi, & Nathan, 2017;Lee, 2017;Mori, Nagamine, Tomita, & Notomi, 2001). Moreover, product detection can be achieved directly within the reaction tube by a diagnostic color change and fluorescence, eliminating the need for electrophoretic techniques and related equipment (Centeno-Cuadros, Abbasi, & Nathan, 2017;Lee, 2017;Mori, Nagamine, Tomita, & Notomi, 2001).…”

mentioning

confidence: 99%

“…This method has also been used for sex determination in a number of organisms, including plants (Fujita et al, 2017;Hsu, Gwo, & Lin, 2012;Shiobara et al, 2011), fish (Hsu, Adiputra, Ohta, & Gwo, 2011), mammals (Khamlor et al, 2015;Nogami, Tsutsumi, Komuro, & Mukoyama, 2008), and birds (Centeno-Cuadros et al, 2017;Centeno-Cuadros, Tella, Delibes, Edelaar, & Carrete, 2018;Chan et al, 2012;Kim et al, 2015). This method has also been used for sex determination in a number of organisms, including plants (Fujita et al, 2017;Hsu, Gwo, & Lin, 2012;Shiobara et al, 2011), fish (Hsu, Adiputra, Ohta, & Gwo, 2011), mammals (Khamlor et al, 2015;Nogami, Tsutsumi, Komuro, & Mukoyama, 2008), and birds (Centeno-Cuadros et al, 2017;Centeno-Cuadros, Tella, Delibes, Edelaar, & Carrete, 2018;Chan et al, 2012;Kim et al, 2015).…”

mentioning

confidence: 99%

“…This method has also been used for sex determination in a number of organisms, including plants (Fujita et al, 2017;Hsu, Gwo, & Lin, 2012;Shiobara et al, 2011), fish (Hsu, Adiputra, Ohta, & Gwo, 2011), mammals (Khamlor et al, 2015;Nogami, Tsutsumi, Komuro, & Mukoyama, 2008), and birds (Centeno-Cuadros et al, 2017;Centeno-Cuadros, Tella, Delibes, Edelaar, & Carrete, 2018;Chan et al, 2012;Kim et al, 2015). Herein, we followed Centeno-Cuadros et al (2017) for generating a LAMP-based protocol to quickly sex juvenile blackcaps (Sylvia atricapilla, Order Passeriformes) in the laboratory or wild. Herein, we followed Centeno-Cuadros et al (2017) for generating a LAMP-based protocol to quickly sex juvenile blackcaps (Sylvia atricapilla, Order Passeriformes) in the laboratory or wild.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Rapid sex determination of a wild passerine species using loop‐mediated isothermal amplification (LAMP)

Koch

Blohm‐Sievers

Liedvogel

2019

Ecology and Evolution

Many bird species are sexually monomorphic and cannot be sexed based on phenotypic traits. Rapid sex determination is often a necessary component of avian studies focusing on behavior, ecology, evolution, and conservation. While PCR‐based methods are the most common technique for molecularly sexing birds in the laboratory, a simpler, faster, and cheaper method has emerged, which can be used in the laboratory, but importantly also in the field. Herein, we used loop‐mediated isothermal amplification (LAMP) for rapid sex determination of blood samples from juvenile European blackcaps, Sylvia atricapilla, sampled in the wild. We designed LAMP primers unique to S. atricapilla based on the sex chromosome‐specific gene, chromo‐helicase‐DNA‐binding protein (CHD), optimized the primers for laboratory and field application, and then used them to test a subset of wild‐caught juvenile blackcaps of unknown gender at the time of capture. Sex determination results were fast and accurate. The advantages of this technique are that it allows researchers to identify the sex of individual birds within hours of sampling and eliminates the need for direct access to a laboratory if implemented at a remote field site. This work adds to the increasing list of available LAMP primers for different bird species and is a new addition within the Passeriformes order.