Annexure- 2

(Declaration to be filled by PhD student)

DECLARATION

Title of the Thesis: “Development of PCR Based Methods for Identification and
Differentiation of Species-Specific Meat”

Submitted for the Ph.D. Degree in the Institute of Bio-Sciences and Technology.

I declare that the presented thesis represents largely my own ideas and work in my own
words. Where others ideas or words have been included, I have adequately cited and listed
in the reference materials. The thesis has been prepared without resorting to plagiarism. I
have adhered to all principles of academic honesty and integrity.

----------------------------

(Signature)
Name of the Student: Mr. Anupam Singh
Registration no.: 201510902000005

i
 Annexure- 3
[Certificate to be filled by Supervisor(s) consider only applicable
Supervisor(s)]
CERTIFICATE -1

This is to certify that the thesis, entitled “Development of PCR Based Methods for
Identification and Differentiation of Species-Specific Meat” submitted by Mr. Anupam
Singh Reg. No 201510902000005 for the award of Degree of Doctor Philosophy in
Institute of Bio-Sciences and Technology of Shri Ramswaroop Memorial University,
Lucknow-Deva Road is a record of authentic work carried out by him under my supervision.

To the best of my knowledge, the matter embodied in this thesis is the original work of the
candidate and has not been submitted for the award of any other degree or diploma of any
university or institution.

Signature of Supervisor: ……………………….

Date: …………………………
Name of Supervisor: Dr. Prachi Bhargava

ii
 Annexure- 4
[Certificate to be filled by the Dean of the Faculty]

CERTIFICATE -2

Certified that the thesis entitled “Development of PCR Based Methods for Identification
and Differentiation of Species-Specific Meat” submitted by Mr. Anupam Singh with Reg.
No. 201510902000005 is a record of bonafide research work carried out by him in the
Institute of Bio-Sciences and Technology, of Shri Ramswaroop Memorial University,
Lucknow-Deva Road, which is of the requisite standard.

__________________
(Signature)
Dean of the Faculty

iii
 ABSTRACT

Meat is used as nutritious food, as it provides a sufficient amount of protein,

vitamins, and minerals to be used for human consumption. In the present time, it is notable
that meat and meat-related products are high in demand, consequent to the rise in the
human population and their disposable income. Nowadays, consumers are concerned about
the adulteration of meat or meat products and request accurate marking; therefore, to
identify the meat correctly, proper checks are needed so that accurate identification can be
made in a short time and the customer can be assured of the right meat.

The presence of species-specific DNA sequences and detecting such sequences has
opened a new era of molecular genetics for identification of respective species using the
assays based on the DNA hybridization probes, PCR or RFLP. In the light of the
knowledge of molecular methods for the species detection, the present examination was
completed to recognize and segregate species-explicit meat viz. rat, chicken, horse, pig,
cow, buffalo, goat, and sheep by developing multiplex PCR assay.

In the present study, two different PCR product amplification approaches have
been used to differentiate between eight different animals (chicken, rat, pig, horse, goat,
sheep, cow, and buffalo) using mitochondrial cyt b and 12S rRNA gene variability.
Mitochondrial Analysis of DNA was the most frequently used DNA because of its highly
conserved sequences in various organism species. In this study, meat samples were
collected from chicken, rat, horse, pig, goat, sheep, cow, and buffalo. Genomic DNA was
isolated from the meat sample, by the Phenol-Chloroform method with slight modification.
For successful achievement of molecular research, particularly for hereditary perspectives,
extraction of high-quality genomic DNA is the prime necessity.

The quality of isolated DNA was visualized on agarose gel electrophoresis and the
concentration of isolated genomic DNA was identified with the help of spectrophotometer.
In the study, two different PCR product amplification approaches using mitochondrial cyt
b and 12S rRNA genes variability were applied. Specific primer sets for cyt b and 12S
rRNA with similar annealing temperatures were designed with Primer3 on the basis of cyt

iv
b (chicken, rat, pig, horse, goat, and sheep) and 12S rRNA (cow and buffalo) gene
.sequences. The specific primers were verified in-silico by SnapGene software. In a 50 µl
volume of final reaction, PCR master mix was prepared and carried out simplex and
multiplex PCR amplification. For confirmation of amplification of PCR product in
respective PCR tubes, run the amplified PCR product with loading dye on 2% agarose gel
containing EtBr (ethidium bromide) at the constant 50V voltage for 50 minutes in 1X TAE
buffer. It was observed that from amplified PCR product of cyt b gene and 12S rRNA gene
by utilizing the protocol of conventional PCR and could be obtained a species-specific
band from isolated genomic DNA from eight meat species. Multiplex PCR was created
and can be used for synchronous recognition of numerous species origin in meat by cyt b,
and 12S rRNA gene-derived species-specific primers.

v
 Acknowledgments
Achievement is not always a success. It is an honest endeavor, persistent effort
to do the best possible under any circumstances. During my Ph.D. research work, I
have been supported and accompanied by many people and is a coveted opportunity
to express my gratitude for them all.

First of all, I express a deep sense of gratitude to the Almighty for giving me a
chance into the wonderful world of science to do something for the welfare of
humanity. I also thank nature for blessing me the favorable conditions.

It is my immense pleasure to express my profound gratitude to my supervisor

Dr. Prachi Bhargava (Associate Professor), IBST, Sri Ramswaroop Memorial
University, Lucknow, for her valuable insight, guidance, optimism and consistent
encouragement throughout the study. The work presented in this thesis would not
have been possible without her excellent guidance and support. Thank you very much,
Madam.

I am grateful to Prof. Sanjeev Kumar Maheshwari, Director, IBST, Sri

Ramswaroop Memorial University, Lucknow, for providing the necessary facilities. I
also extend my thanks to all the other faculty members of IBST for their
encouragement.

I express my sincere thanks to Prof. Krishna Srivastava, INSH, Sri

Ramswaroop Memorial University, Lucknow, and Dr. Divya Gupta (Associate
Professor), IBST, Sri Ramswaroop Memorial University, Lucknow for providing the
construct for the present study.

I express my heartfelt and sincere thanks to Dr. Mahesh Basantani (Professor

and Dean), FOBS, IBST, Sri Ramswaroop Memorial University, Lucknow for his
encouragement, and immense support throughout the work.

I am grateful to Dr. Mukul Mishra, Director Research, Sri Ramswaroop

Memorial University, Lucknow, for his suggestions in the present study.

I express my deep-felt thanks to my colleagues, Mr. Ashish Kumar Rai

(Assistant Professor, DBT, BIET, Lucknow), Mr. Gyan Datta Tripathi (Assistant
Professor, DBT, BIET, Lucknow), for their immense support and ever ready help. The
affectionate cooperation and lively company of Mr. Veevekanand Srivastava, Ms.
Neha Gupta, and Mr. Kuldeep Srivastava during my crucial period of work is
unforgettable. It is my pleasure to acknowledge the helping hand and lively company
of Mr. Latafat Chaudhary and Asmat. My heartiest thanks to all of them.
vi
 I express my sincere thanks to the technical and administrative staff at Sri
Ramswaroop Memorial University, Lucknow, for helping in every possible way.

I become speechless when it comes to the time to acknowledge my family

because no words can address my sincere feeling towards all of them. My parents and
my in-laws did a lot for me. I also dedicate this Ph.D. thesis to my lovely son Shaurya,
who is the pride and joy of my life. I love you more than anything, and I appreciate all
your patience and support during daddy's Ph.D. studies. Dr. Ragini, my wife, I owe
you a big thank for your unconditional love, endless support, and encouragement
through this time.

I acknowledge the financial support of SERB, New Delhi.

Finally, I acknowledge Sri Ramswaroop Memorial University, Lucknow, for

providing me a platform from where I can scale the highest heights in my carrier.

Thank you all.

(Anupam Singh)

vii
 LIST OF ABBREVIATIONS
% Percent

μg Microgram

μl Microliter

AFLP Amplified Fragment Length Polymorphism

BLAST Basic Local Alignment Search Tool

BP Base pair

BSE Bovine spongiform encephalopathy

PCR Polymerase Chain Reaction

pg Picogram

cyt Cytochrome

DNA Deoxy ribonucleic acid

DNAase Deoxyribonuclease

dNTP Deoxy nucleotide triphosphate

dsDNA Double stranded DNA

EDTA Ethylene diamine tetra acetate

Fig Figure

RFLP Restriction Fragment Length Polymorphism

LINE Long Interspersed Nuclear Elements

M Molar

MBM Meat and Bone-Meal

mg Milligram (s)

Min Minute

mM Milli molar

mRNA Messenger RNA

viii
mt Mitochondria

mt DNA Mitochondrial Deoxy ribonucleic acid

MW Molecular weight

NFW Nuclease Free Water

ng Nanogram (s)

OD Optical Density

RNA Ribonucleic acid

SDS-PAGE Sodium dodecyl sulphate-Polyacrylamide

Gel Electrophoresis

TAE Tris Acetate EDTA

TE Tris EDTA

UV Ultra Violet

w/v Weight / volume

ix
 TABLE OF CONTENTS

CHAPTER TITLE PAGE

NO.

1 INTRODUCTION 1-6

2 REVIEW OF LITERATURE 7-27

2.1 Food authenticity and its importance 7
2.2 History of food authentication 8
2.3 Techniques for meat authentication 9
2.3.1 Organoleptic techniques 10
2.3.2 Chemical methods 11
2.3.3 Anatomical techniques 11
2.3.4 Electrophoresis method 11
2.3.5 Poly acryl amide gel electrophoresis (PAGE) 12
2.3.6 SDS-PAGE (Sodium Dodecyl Sulphate PAGE) 12
2.3.7 IEF (Isoelectric focusing) 13
2.3.8 2-DE (Two-dimensional electrophoresis) 13
2.3.9 Capillary electrophoresis 14
2.3.10 Chromatography 14
2.3.11 Spectroscopy 15
2.3.12 Immunological method 15
2.3.13 Biochip 17
2.3.14 Molecular approaches based on DNA for meat species
detection 17
2.3.14.1 DNA targets to identify meat 17
2.3.14.1.1 Nuclear DNA 17
2.3.14.1.2 Mitochondrial DNA 18
2.3.14.2 Different DNA based techniques for meat identification 19

x
 2.3.14.2.1 DNA hybridization 19
2.3.14.2.2 PCR based techniques 20
2.3.14.2.2.1 Random amplified polymorphic DNA finger-
printing (RAPD) 20
2.3.14.2.2.2 Micro-satellite markers 21
2.3.14.2.2.3 RFLP (Restriction Fragment Length
Polymorphism) 21
2.3.14.2.2.3 FINS (Forensically Informative Nucleotide
Sequencing) 23
2.3.14.2.2.4 RT-PCR (Real-Time PCR) 24
2.3.14.2.2.5 Species-Specific PCR 25
2.3.14.2.2.6 Multiplex PCR 26

3 MATERIALS AND METHODS 28-36

3.1 Materials 28
3.1.1 Meat samples collection 28
3.1.2 Chemicals 28
3.1.2.1 Chemicals for DNA isolation 28
3.1.2.2 Chemicals for PCR 29
3.1.2.3 Chemicals for gel electrophoresis 29
3.1.3 Glassware and plasticware 29
3.1.4 Equipments 29
3.2 DNA Extraction from meat sample 30
3.3 PCR S amplification 31
3.3.1 DNA template 31
3.3.2 Primer design, synthesis and validation 31
3.3.3 Preparation of reaction mixture for PCR 32
3.3.4 PCR optimization 35
3.3.4.1 Primer concentration 35
3.3.4.2 Denaturation temperature 35

xi
 3.3.4.3 Annealing temperature 35
3.3.5 Protocols for PCR amplification 35
3.4 Confirmation of PCR amplification 36

4 RESULTS 37-54
4.1 Genomic DNA isolation 37
4.2 Quantitative estimation of DNA 38
4.3 Primer validation 38
4.3.1 Primer sequence and target region on cytochrome b gene 38
4.3.2 Primer sequence and target region on 12S rRNA gene 40
4.3.3 In-silico validation of designed primers (cyt b gene) 41
4.3.4 In-silico validation of designed primers (12S rRNA gene) 44
4.4 PCR Amplification 45
4.4.1 Simplex/Conventional PCR 45
4.4.2 Multiplex PCR 47
4.5 Detection limit of DNA in PCR 49

5 DISCUSSION 55-60
5.1 Protein based methods 55
5.2 DNA based methods 56
5.3 Amplification of cyt b gene by conventional PCR 55
5.4 Amplification of 12S r RNA by conventional PCR 58
5.5 Cytochrome b gene and 12S r RNA gene amplification by
multiplex PCR 59

6 SUMMARY AND CONCLUSION 61-63

REFERENCES 64-71

APPENDICES 72-74

xii
 LIST OF TABLES
Table TITLE OF TABLES Page
No. No.

3.1 Primer sequences amplifying cyt b gene 32

3.2 Primer sequences amplifying 12S rRNA gene 32

3.3 Details of PCR components used for simplex reactions 33

3.4 For duplex reaction (Cow and Buffalo) details of PCR components 33

3.5 For multiplex PCR reaction (Buffalo, Cow, Goat and Sheep) 34
details of PCR components

3.6 For multiplex PCR reactions (Chicken, Pig, Horse and Rat) 34
details of PCR components

3.7 For amplification of cyt b gene, details of PCR cycle 36

3.8 For amplification of 12S rRNA gene, details of PCR cycle 36

4.1 Quantitative estimation of DNA 38

4.2 DNA detection limit 54

xiii
 LIST OF FIGURES

Fig. No. TITLE OF FIGURES Page

No.

1.1 Feed required to produce one kilogram of meat 3

2.1 Methods for specific meat species identification 10

4.1 Agarose gel Electrophoresis of genomic DNA isolated from meat 37

samples.

4.2 Primer sequence and target region on cytochrome b gene. 39

4.3 Primer sequence and target region on 12S rRNA gene. 40

4.4 In-silico validation of designed primers for chicken 41

4.5 In-silico validation of designed primers for rat 42

4.6 In-silico validation of designed primers for pig 42

4.7 In-silico validation of designed primers for horse 43

4.8 In-silico validation of designed primers for goat 43

4.9 In-silico validation of designed primers for sheep 44

4.10 In-silico validation of designed primers for buffalo 44

4.11 In-silico validation of designed primers for cow 45

xiv
4.12 Visualization of amplified PCR products with simplex PCR for the 46
fragments of cow, Buffalo, Goat and Sheep

4.13 Visualization of amplified PCR products with Simplex PCR for the 46
fragments of Chicken, Pig, Horse and Rat (cyt b gene)

4.14 Visualization of amplified PCR products with Multiplex PCR for the 48
fragments of Cow, Buffalo, Goat, and Sheep 12S rRNA and cyt b gene

4.15 Visualization of amplified PCR products with Multiplex PCR for the 49
fragments of Chicken, Pig, Horse and Rat (cyt b gene)

4.16 The sensitivity of the PCR for Goat 50

4.17 The sensitivity of the PCR for Chicken 50

4.18 The sensitivity of the PCR for Buffalo 51

4.19 The sensitivity of the PCR for Sheep 51

4.20 The sensitivity of the PCR for Pig 52

4.21 The sensitivity of the PCR for Horse 52

4.22 The sensitivity of the PCR for Cow 53

4.23 The sensitivity of the PCR for Rat 53

xv
xvi