Introduction :-

1)This research discussed the potential applications of DNA barcoding in routine mean inspection in
Phillipines. Developing mini-barcode primer sets to enhanced the utility of conventional techniques
is critical in adopting DNA barcoding technology as a robust tool for the routine inspection of meat
sold commercially, including those intended for the halal meat industry.

2) DNA based technique for widly used for species identification.In presence research cooked,meat
&bone were analysed successfully on the basis of cytochrome oxidase sub unit,I (coi) marker.the
query sequence obtained was compared to that of the reference in NCBI gene bank database &bold
database.

3)In this study sensitive & specific method multiplex nested PCR was applied to identify commercial
meat species dry blood was use as an alternative DNA sources for detection for sensitivity was in has
biprimers specifically designed to encompass the mitochondrial cytochrome b & NADH
dehydrogenase 5/6 gene the specificity & sensitivity of multiplex PCR system.where tested different
length of specific nsted PCR products were detected to be 350,570,750&1000 bp for chiken
pig,cow,& crocodile respectively the system allow detection with as little as found nanogram of DNA
from either meat or blood sample. Detection sencitivity of individual species was improve, enabling
the detection of DNA. With as a little 1 as picogram.

4)Avijit Ghosh present study has been conducted in view of ascertaining the species of origin from
fully cooked meat that was confiscated from a restaurant at murshidabad district of West Bengal in
India & suspected to be originated from a greater short-toed Lark.there was no intact morphological
identity,&the owner of the restaurant was booked under the charges of Indian wildlife (protection)
Act 1972 towards killing & selling meat of protected species.

5) A DNA barcoding approach based on DNA sequence from the co1 gene,we were able to show
within the one day that the animal skin was actually the skin of cow.(Bos indicus) that had been
altered to appear as a tiger skin. Although DNA barcoding is not a novel application of method for
species identification.the rapid turnaround time needed to obtained conclusive results in such case
is an important factor that brings novelty to this method in forensic science.

6) Drug trafficking is one of most profitable forms of crime & in the past few decades.The presence
of cocaine an and / or its isomers was analysed by Scott test & modified Scott test; and confirm by
LC-MS / MS.cocaine hed positive results for Scott test performed directly on the sample & for the
modified Scott test.perfomed with the contaminated stunning water.The analyses performed by LC-
MS/MS with samples of gills,entrails & flesh showed a single with the same MRM transition of
cocaine,but in a different retention time and with different relatives ions intensities, inferring the
presence of pseudo cocaine instead of cocaine.
7) le explores how forensic technologies are and/or could be deployed to counter and prevent
crimes against the environment. Our specific focus is littering and the application of DNA analysis.
Recent years have seen a wide range of technological and social innovations in regards
environmental crime prevention, detection, investigation, and prosecution. These have included, for
example, the use of microchips in wood and seafood products to track their movements from
country of origin to country of destination, satellite and drone surveillance of changing land use and
land clearing, chemical composition analysis of oil spills, and DNA identification of salmon and
abalone for the purpose of criminal prosecution.

8)The helmeted hornbill is unique in that it is the only hornbill species that grows a large beak with a
solid casque that is composed of keratin – the same substance that makes hair, horns, hoofs and
teeth in mammals. The casque, which is typically pale orangey yellow with a thin outer red layer, is
so heavy that, along with the skull, can account for around 11% of the bird’s weight. The bird uses its
bill as a hammer and sometimes as a battering display by males in head-to-head aerial jousting
during the breeding season, and as a weighted tool to dig out animals from rotting logs by both
sexes.

9) The processing of FTA cards bearing blood samples starts with the washing of cell debris and
heme inhibitors present in blood with the help of a purifying reagent and leaving DNA immobilized
within the FTA matrix. With the classic FTA purification process, once the punches drawn from the
blood spots on the FTA card become clear they can be used directly for PCR amplification.It has been
often observed that the medical officers and investigating Officers try to fillthe entire circle(s)
marked on the FTAcard with blood.In The process, the card gets overloaded with blood.

10)The illegal wildlife trade is a significant threat to many extant wild animal species worldwide. To
combat the illicit wildlife trade, species-level identification of seized wildlife parts or products is
required for the legal prosecution of the accused/s in a court of law.Almost all wild bovid species in
trade worldwide serve as big and small game animals for their magnificent horn trophies, head
mounts, skin/fur,bones, meat and other body parts. Horns, the characteristic feature of species in
the family Bovidae, have made these species an important big game animal throughout the world
and have served as a key constituent in traditional medicines worldwide since time immemorial.

 
Aim :-

           Technique for meat inspection: An forensic application.

Objective :-

. Incorporating animals forensic in routine meat inspection in the Phillipines.

. DNA barcoding of meat and bone samples of suspected endangered species: A case study

. Identification of species (meat and blood samples) using nested PCR analysis of mitochondrial DNA.

. Wildlife forensic in voiding false offences: A case study to deal with unidentified cooked meat.

. DNA barcode based identification of suspected tiger skin: A case to resolve a mimicry.

. Frozen fish & (pseudo) cocaine?

A case report

. Forensic science and environmental offences: Litter, DNA analysis  and surveillance.

. DNA recovery and analysis from helmeted hornbill (Rhinoplax vigil) Casques and its potential
application in wildlife law enforcement.

. Development and evaluation of a novel DNA purification buffer and protocols For blood samples on
FTA cards.

. A pioneering method to identify bovine horn trophy: A combined Morphometric and DNA-based
approach in wildlife forensics.
Literature review :-

Khedkar et.al., (2019) analysed suspected (Panthera tigris)animal skin which was later on

Found that it is of Bos indicus. Amplified PCR technique was used for DNA sequencing and

Compared with the already existing NCBI gene bank. The researcher confused with the stripe

Pattern painted with dye on the skin which was similar with the Panthera tigris. Here DNA

Sequencing resolved the case of mimicry.

Data collection :-

1) Data obtained from using DNA barcoding for the identifying number of species use in
research out of 85 unidentified pangollin car cases,73 were identified in phillipines.
Common meat and adult adulterated meat. In Egypt 6 & 2 sample. Were positive from
doncky,horse,meat. Out of 96 beef sample.
2)
3) DNA from meat sample, bone, Spinal cord, blood detected on wooden piece and gunny bag
and cooked meat sample was extracted using manual extractions protocol.
4)

3)      In data collection Fresh chicken, cow, pig and crocodile meats were collected from local market
in Thailand. Blood samples were directly withdrawn from live animals by using 0.5 M ethylene
diamine tetra-acetate (EDTA) solution as an anti-coagulant. Blood mixture was stored in sterile 50 ml
centrifuge tube, subsequently freeze dried to obtain dried blood powder, and stored at 4°C until
used.

5) A sample of fully cooked meat was seized from a restaurant Located in Murshidabad
district of West Bengal, India by the Officials of Eastern Regional Office – Wildlife Crime
Control Bureau (WCCB), West Bengal.Based on the local information gathered by the
WCCB, the offence was booked under the charges of the Wildlife (Protection) Act, 1972
suspecting the seized meat was of STL origin since there have been reports of a massive
catch of STL in last few years for local consumption. The seized material in the form of fully
cooked meat seasoned with spices and Condiments was sent to Zoological Survey of India,
Kolkata for Ascertaining the species of origin.
6)

5)      To resolve the issue, the skin wassent to ourlaboratory for DNA-based identification from our
initial analysis, the obtained skin sample appeared to be highly processed with chemicals/herbs and
dyes. For DNA testing,we otained pieces around 0.5 cm2 (in triplicate) and 10 hairs with follicles
from the skin. These were washed with 0.8% saline several times to remove the curatives used to
preserve the skin. A final wash absolute ethanol was given.

7) 8 test samples of frozen fish were randomly selected and prepared for Chromatographic
analyses, each sample from a questioned material. Gills Were removed from the samples 1
to 6. Sample 7 was used for the Examination of entrails and sample 8 for the analysis
offlesh.
8)

7)      DNA is unintentionally discarded by shedding bodily matter such asSkin cells, sweat, and hair,
and through bodily fluids, such as saliva,Semen, urine, hair and blood, even in trace amounts. The
potential Therefore exists for a range of DNA analysis techniques to be used on DNA obtained from
litter. As DNA is found in cells and contains general information common amongst people as well as
personal information,Including identification markers (unique to an individual except in Identical
twins) and ancestral history [22], the potential to exploit litters as a source of DNA for analysis
warrants consideration.
 9) In this study, casques from 9 helmeted hornbills were tested, 2 Specimens were obtained
from the Thailand Hornbill Project (THP) that Had been donated by the local villagers from
the Southern of Thailand,And 7 specimens were from confiscated cases, taken from the
Museum of Confiscated Wild Animals of Department of National Parks, Wildlife and Plant
conservation (DNP), Bangkok, Thailand. The sources of material.
10)

9)      More than 200 FTA cards submitted to State Forensic Sciences Laboratory Junga, Shimla by
Medical officers and investigating officers For DNA analysis were evaluated. A visual inspection
clearly showed that All these samples were overloaded with blood. The FTA cards were Processed
with conventional purification reagents and modified Purification process with the newly designed
buffer system. In additions 100 FTA cards, that were not overloaded with excess blood samples
Were Also subjected to purification with the novel buffer based protocol and Subsequent PCR and
Short Tandem Repeat (STR) analysis.

11) We used a horn trophy with metal coverings on its tip and at the base, Suspected to be
from a wild water buffalo. This trophy horn was seized By the authorities in Delhi, India, in
April 2014 and sent to the Wildlife Institute of India, Dehradun, for species identification.
12)

Conclusion :-

1) DNA barcoding & mini barcoding technique for reliable identification of row meat & meat
product for rapid & cost effective methods of meat species identification during inspection.
The phillipines should strictly implement meat product authenticity testing with proper
labeling, branching & packing to enable consumer to obtain accurate information & ensure
traceability of animal meat product.
2)

3) In this case, identification was done with the help of COI gene. This method is reliable as it
showed versability in its ability to use a single conserved primer pair to accurately identify.
If con specific sequence were available in database.
4)

 
3)      To determine the specificity of the primer set, the Universal primer designed based on the
conserved region Of the mitochondrial Cyt b and ND5/6 gene, was initially Used to amplify in the
first PCR reaction. Amplicons with Approximately 2,000 bp were detected in pig, cow, and Crocodile
DNA, whereas 1,400 bp amplicon was only Observed in chicken DNA. To increase detection
determined according to the predicted species-specific Amplicons as follows: 350 bp for chicken, 570
bp for pig,750 bp for cow and 1,000 bp for crocodile Results showed that the species specific nested-
PCR Primers can detect DNA at 0.001, 0.01, 0.5, and Result Level of cow, crocodile, chicken, and pig
meats, respectively. These nested-primer sets could also detect at 0.001, 0.05, 0.5, and 5 ng level of
cow, pig, crocodile, And chicken blood DNA templates as well

Or blood was detected to correspond to each animal Species without cross-contamination.

5) Yielded sequence lengths of the confiscated material for both genes Were 287 bp (Cytb)
and 360 bp (12S rRNA). The percent similarity of confiscated material with suspected STL
was 79% only for Cytochrome b Gene andno datawas available for12S rRNAgene sequence
in the database. A99%similarity of the generated sequences from confiscated material with
common quail (Coturnix coturnix) for both the genes was found. The Generated sequences
in this study were submitted to GenBank/NCBI Database withAccession nos. MH423749
and MH423716.Thus,the results Indicated that confiscated material was a Common quail
and not the STL.
6)

5)      The DNA sequence obtained (650 bp) was searched for similar Sequences on the BOLD system
and by using the BLAST program of NCBI’sGenbank . Based on the sequence similarities obtained,
the Confiscated skin was shown to belong to the Indian cow (Bos indicus). Absolutely reasonable
match was observed with in-house reference Sequences derived from Panthera tigris. Phylogenetic
analysis using the Program MEGA10.0 [12] also revealed no correlation between COI Sequences of
Panthera tigris and the sequence from the present study.Over all this work supports the idea of
using DNA in forensics Applications for identification of unknown materials, and that this Approach
should be used to the fullest extent possible to combat illegal Trade in wildlife. Specifically in the
case presented here, a rapid Turnaround time wasrequired because the investigating officer needed
to Present the analysis of the evidence in a court of law. Using the DNABarcoding approach, we were
able to submit reports within a day fromReceipt of samples. Over time, this approach will also
become even more Powerful as information from more species and genes are added to the
Databases used to provide reference information.

 
 7) After the analyses of cocaine/pseudo cocaine in the fishes,a short stability evaluation
showed the lack of reproducibility of the candidate pseudo cocaine,with high signal of
degradation after 12hr. The confirmation of the compund present in the extract by
analytical standard would be valuable,since a singnal was detected with different retention
& time & different relatives ions intensities,but with the same fragmentation pattern of
cocaine as it was mentioned above, however the analytical standard of pseudo cocaine was
not available. During the method development & analysis to confirm these results.
8)

7)      Litter is a pervasive problem impacting the environment. This article Has considered how DNA
analysis may be used in various ways on DNA Obtained from litter. While the potential of DNA
analysis reflects ad-Vances in science and technology, and changes in policing to a more Intelligence-
led approach, various ethical issues arise from genetic sur-Veillance that encompasses litter as a
source of DNA for analysis. Two Issues in particular stand out. The first is the linking of methods (e.g.,
DNA phenotyping) with the transgression (i.e., littering) in ways that Appear to be disproportionate
to the gravity of the offence and that may, Unintentionally, institutionalise social bias (e.g., the
targeting by the State of particular population groups, such as young people or people From ethnic
minority backgrounds). Second, and related to this, the ease Of application of the technologies (e.g.,
the growing availability and Simplicity of sampling, and the advent of big data) also means that there
Is a level of potential surveillance and intrusion that has significant Implications for privacy, human
rights and individual autonomy.