Introduction Snakes are experiencing worldwide population declines. Consequently, researchers require effective and reliable methods for detecting the presence of threatened snake species in critical habitats. Snakes, however, can be challenging to reliably detect during surveys due to their secretive habits and cryptic lifestyle. Environmental DNA (eDNA) assays are increasingly being used to aid ecological studies for the detection and discernment of target species populations. eastern pine snake ( Pituophis melanoleucus ) are secretive, semi-fossorial snakes found in southeastern United States and are considered a species of conservation concern in all states where they occur. At present, no species-specific primers exist for the detection of the eastern pine snake from eDNA. Methods We developed a quantitative PCR (qPCR) and end-point PCR assay using novel eastern pine snake-specific primers targeting the NADH dehydrogenase subunit 4 ( ND4 ) gene region. The developed primers and the assay conditions were validated for specificity, sensitivity, and application to eDNA for the detection of eastern pine snake from field samples. Results Our novel assay was determined to be specific and sensitive for the detection of eastern pine snake when tested against DNA from tissues or shed skins of heterospecific species and when applied to eDNA from the soil samples obtained from field sites. Discussion The developed primer set and eDNA assay provide an efficient, noninvasive tool to complement traditional survey techniques (i.e., camera traps and visual surveys) for detecting the presence of eastern pine snake from soil samples.
Kinavey et al. (Wed,) studied this question.