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

Abstract

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.

Comparison of different DNA preparatory methods for development of a universal direct PCR-RFLP workflow for identification of meat origin in food products

“…As horse meat has also been part of food adulteration scandal and a lot of public mistrust in the past (Premanandh, 2013), it was preferred that the developed assay be capable to analyze horse meat DNA. Our buffer selection coincides with most of the recent studies that have adapted alkaline lysis for DNA extraction (Koch et al, 2019;Martincová & Aghová, 2020;Bui et al, 2021) as they have used AL 3 as their lysis buffer. The developed fast extraction protocol of DNA might be helpful for quicker PCR-based identification of meat species in testing laboratories.…”

Optimization and application of loop‐mediated isothermal amplification technique for sex identification in red‐whiskered bulbul (Pycnonotus jocosus)

“…DNA amplification from sex chromosomes by PCR has been widely reported, and the specific method has been shown to be highly effective (Griffiths et al, 1998). However, due to time-consuming protocols and the requirement of specialized laboratory instruments, PCR is not suitable for rapid application and efficient study in field-scale (Centeno-Cuadros et al, 2016Chan et al, 2012;Koch et al, 2019;Notomi et al, 2000). In this study, sex F I G U R E 8 Sensitivity of post-LAMP analysis method compared with electrophoresis using DNA concentration between 10 3 -10 −6 ng were used for sex identification (Chan et al, 2012).…”

“…DNA amplification from sex chromosomes by PCR has been widely reported, and the specific method has been shown to be highly effective (Griffiths et al, 1998). However, due to time‐consuming protocols and the requirement of specialized laboratory instruments, PCR is not suitable for rapid application and efficient study in field‐scale (Centeno‐Cuadros et al, 2016, 2017; Chan et al, 2012; Koch et al, 2019; Notomi et al, 2000). In this study, sex identification of the red‐whiskered bulbul by the LAMP technique was developed by identifying the CHD gene of sex chromosomes Z and W. The CHD‐W reaction distinguishes between males and females because the CHD‐W gene is only found in female birds.…”

Development of a Rapid Sex Identification Method for Newborn Pigeons Using Recombinase Polymerase Amplification and a Lateral-Flow Dipstick on Farm

“…Isothermal amplification methods are suitable for less well-equipped locations. Among existing isothermal amplification methods, loop-mediated isothermal amplification (LAMP) represents a popular method that was developed for the purposes of bird sexing [ 14 , 15 , 16 ]. The recombinase polymerase amplification (RPA), another isothermal technique, can also be used to amplify specific DNA within a single temperature range (37–42 °C) [ 17 ].…”