Page 41 - Veritas Vol 3, Issue 2

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CASE DETAILS:

A restaurant located in the Murshidabad district of West Bengal, India, was
accused of using poached meat, and so the officials of the Eastern Regional

Office of the Wildlife Crime Control Bureau (WCCB), West Bengal, seized a
sample of fully cooked meat from the restaurant. The suspect (the restaurant

owner) was booked under the charges of the Wildlife Protection Act, 1972,
suspecting the seized meat was of STL origin (a species of target concern)

since the officials received reports of illegal poaching of STL in the last few
years for local consumption. The fully cooked meat sample was sent to the

Zoological Survey of India, Kolkata, for species identification.

STEPS INVOLVED:

The meat is washed with 1× sterile phosphate buffered saline (PBS)

overnight to remove all the remains of spices and condiments.
A small pinch of flesh from the innermost layer of the sample is taken.

Isolation of genomic DNA is carried out through the QiAmp DNA-
investigator kit (Qiagen Germany).

Universal primer sets of Cytochrome b and 12S rRNA genes are used to
amplify approximately 400 bp fragments of both mitochondrial genes.

Sanger sequencing, also known as chain-termination sequencing, which
is a method used to determine the sequence of nucleotides in a DNA

molecule, was carried out on the Genetic Analyzer 3730. Sequence
quality was checked and validated using sequencer V4.7.

Homologous sequences were downloaded from the NCBI/BLAST
database, and multiple sequence alignment was performed by the

CLUSTALW algorithm in MegaX.

Best fit models were identified using Bayesian Information Centre (BIC)
values for all the downloaded and generated sequences, and maximum
likelihood trees were constructed in MEGA X using the Hasegawa-

Kishino-Yano model (HKY + G) and Tamura-Nei model with a gamma

distribution for cytochrome b and 12S rRNA gene sequences.
The generated sequences were submitted to the GENBANK/NCBI

database with accession numbers MH423749 and MH423716.

VERITAS VOLUME: 3, ISSUE: 2 WRITER: SRI INEYA K