A novel multiplex PCR assay for the identification of Indian crocodiles

Abstract

Illegal hunting has been a major threat for the survival of wildlife fauna, including the three crocodile species that India harbours: Crocodylus palustris, Crocodylus porosus and Gavialis gangeticus. Although law prevents trade on these species, illicit hunting for trade continues to threaten the survival of these endangered species; conservation strategies therefore require a rapid molecular identification technique for Indian crocodiles. A multiplex polymerase chain reaction (PCR) assay with species-specific primers, considered as one of the most effective molecular techniques, is described herein. The primers were designed to yield species-specific sized amplicons. The assay discriminates the three Indian crocodile species unambiguously within a short time period using only simple agarose gel electrophoresis. We recommend this multiplex PCR assay to be used in the identification of Indian crocodile species.

Complete mitochondrial genome sequences of three Crocodylus species and their comparison within the Order Crocodylia

“…Besides the evolutionary perspective, the polymorphisms in the mitochondrial genomes have been utilized for various ecological studies (Avise, 1995;Valentini et al, 2009). The forensic utility is another aspect where the mitochondrial genome sequences have proved useful for species identification and thereby are a valuable tool for the conservation of endangered species (Teletchea et al, 2005;Dubey et al, 2009Dubey et al, , 2010Meganathan et al, 2009aMeganathan et al, , 2010a. Thus, the generation of mitochondrial genome sequences is useful for phylogenetic analyses and also for informing conservation efforts.…”

Rapid species identification of pygmy rabbits (Brachylagus idahoensis) from faecal pellet DNA

“…Species identification using DNA isolated from faecal samples has been accomplished for several mammalian species (Waits & Paetkau 2005). Species identification is achieved by direct sequencing of mitochondrial DNA (Farrell et al 2000;Adams et al 2003), restriction enzyme analysis (Paxinos et al 1997;Kovach et al 2003), or multiplex fragment analysis using species-specific primers (Dalén et al 2004;Dubey et al 2009;Meganathan et al 2010;Stewart et al 2010). Direct sequencing and restriction enzyme analysis can be costly and time consuming.…”

Validation of a Multiplex PCR Assay for the Forensic Identification of Indian Crocodiles*

“…The multiplex PCR was carried out for all the samples using the procedure described in the study by Meganathan et al. (7). All the reactions contain 10 ng of DNA templates unless otherwise mentioned.…”

“…The multiplex reactions were carried out in 25.0-lL reaction volumes in duplicates on GeneAmp Ò 9700 thermal cycler (Applied Biosystems, Foster City, CA). The PCR ingredients and the cycling conditions are described in the study by Meganathan et al (7), and all the ingredients and PCR conditions were kept the same for all the reactions unless otherwise mentioned. The PCR products were checked in 2.5% agarose gel with 0.5 lg ⁄ mL of ethidium bromide stain.…”

“…Therefore, a simple multiplex PCR assay was developed based on cytochrome b (cyt b ) gene, in which the species‐specific forward primers: MUG for C. palustris , SAL for C. porosus , GHA for G. gangeticus and a universal reverse primer, UNI were designed to yield specific‐sized PCR products for each species. This novel multiplex PCR aids in quick and unambiguous identification of Indian crocodile species (7). However, judicious use of this technique in forensic cases requires a validation study to be completed before it can be implemented in the real forensic scenario (8).…”