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

Abstract

A dependable and efficient wildlife species identification system is essential for swift dispensation of the justice linking wildlife crimes. Development of molecular techniques is befitting the need of the time. The forensic laboratories often receive highly ill-treated samples for identification purposes, and thus, validation of any novel methodology is necessary for forensic usage. We validate a novel multiplex polymerase chain reaction assay, developed at this laboratory for the forensic identification of three Indian crocodiles, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, following the guidelines of Scientific Working Group on DNA Analysis Methods. The multiplex PCR was tested for its specificity, reproducibility, sensitivity, and stability. This study also includes the samples treated with various chemical substances and exposed to various environmental regimes. The result of this validation study promises this technique to be an efficient identification tool for Indian crocodiles and therefore is recommended for forensic purposes.

Double gene targeting multiplex PCR-RFLP detects Crocodylus porosus in chicken meatball and traditional medicine

“…Earlier, we have scientifically proven that the stability of the PCR assay under extensive processing atmosphere largely depends on the amplicon sizes; longer targets break down before the shorter ones. [26] This study carefully addressed this point and kept amplicon lengths of 77 bp and 127 bp; far shorter than previous amplicon sizes of 628 bp and 780 bp; [18,19] 373 bp, 486 bp, and 578 bp; [20,21] 1000 bp and 2000 bp [17] and 600 bp and 690 bp. [22] Moreover, double gene sites were used as targets for each species to complement a potential missing target.…”

“…To the best of our knowledge, no other studies have tested the double gene mPCR assay for crocodile under food processing conditions such as boiling, extreme autoclaving (2.5 h) and microwave cooking (700 W for 30 min). Previous research by Meganathan and coworkers [21] reported sensitivity up to 10 pg in highly putrefied crocodile samples, tissues, and blood, but the sensitivity under the processed sample remained inconclusive. In this research, double gene targeted species in model meatballs were amplified at all levels of adulteration including the 10% autoclaved model chicken meatball samples (Fig.…”

“…As authentication is concerned, six PCR assays [17][18][19][20][21][22] have been proposed for crocodile identification. Two of which are conventional PCR, [18,22] one PCR-RFLP [19] and the other three are multiplex PCR assays.…”

“…Two of which are conventional PCR, [18,22] one PCR-RFLP [19] and the other three are multiplex PCR assays. [17,20,21] However, the documented methods are based on very long amplicon-lengths (373 bp-2000 bp) that are not so stable under extensive processing of food, pharmaceuticals, and cosmetics. Although the authors have claimed the stability of their assays in putrefied samples, numerous recently published reports [23][24][25] could not support the sustainability of so long ampliconbiomarkers, especially under the states of decomposition.…”

Identification of Indian Crocodile Species Through DNA Barcodes

“…Crocodile conservation management in India sought for development of novel identification methods for forensic analyses. In this context, we have previously developed some molecular techniques to identify the seized sample derived from Indian crocodiles . Describing the utility of other widely used molecular methods for these species is extremely important to help in the selection of appropriate technique in forensic application.…”