PCR Applications in Identification of Saliva Samples ...€¦ · 12 Cross tabs between PCR method...

PCR Applications in

Identification of Saliva Samples

Exposed to Different Conditions

(Streptococci Detection Based)

Thesis Submitted for partial fulfillment of the MD Degree

in

Forensic Medicine And Clinical Toxicology

By

Mona Mohamed Aly Mohamed M.B., B.Ch. M.Sc.

ASSISTANT LECTURER OF FORENSIC MEDICINE AND CLINICAL TOXICOLOGY

Faculty of Medicine, Cairo University

Supervised by

Prof. Dr. Dina Ali Shoukry PROFESSOR OF FORENSIC MEDICINE AND CLINICAL TOXICOLOGY

Faculty of Medicine – Cairo University

Prof. Dr. Hala Saied Zaghloul PROFESSOR OF FORENSIC MEDICINE AND CLINICAL TOXICOLOGY


Prof. Dr. Laila Ahmed Rashed PROFESSOR OF BIOCHEMISTRY


Dr. Mona Gamal Eldin Nada LECTURER OF MICROBIOLOGY AND IMMUNITY


Faculty of Medicine

Cairo University

2012

AcknowledgementAcknowledgement

First and foremost I feel always indebted to God, the great creator and the

source of all man’s knowledge.

No words can fulfill my deep respects and my feeling of gratitude to Prof.

Dr. Dina Aly Shoukry Professor of Forensic Medicine and Clinical Toxicology,

Faculty of medicine, Cairo University, for her great support, motherly attitude,

sincere encouragement, valuable guidance, kind supervision.

I would like to express my deepest gratitude to Prof. Dr. Hala Saied

Zaghloul Professor of Forensic Medicine and Clinical Toxicology,Cairo

University, for her sincere supervision, guidance, follow up and support.

My deep gratitude to Prof. Dr. Laila Ahmed Rashed Professor of

Biochemistry, Cairo University, for her meticulous supervision, kind support,

reliable advice and kind help in every step of this work.

Great thanks to Dr. Mona Gamal Eldin Nada Lecturer of Microbiology

and immunity for her support.

My deep gratitude to My Family especially My Parents for their great and

kind support and encouragement.

Last, but not least, my deep thanks to all my professors and my colleagues.

List of contents

List of contents

I

Title Page number

Introduction 1-2

Chapter 1: Saliva Composition and Functions: A

comprehensive Review

3-10

Chapter 2: Saliva as a biological Evidence 11-14

Chapter 3: Methods of Saliva Detection 15-26

Chapter 4: Methods of DNA Extraction from Saliva 27-36

Chapter 5: PCR (The polymerase chain reaction) 37-47

Chapter 6: Role of PCR in Saliva Identification 48-52

Materials&Methods 53-61

List of contents

List of contents

II

Results 62-83

Discussion 84-94

Conclusion &recommendations 95

Summary 96-97

References 98-108

الملخص العربى

List of abbreviationList of abbreviation

III

(ATP) Adenosine triphosphate

(AUC) Area under the curve

(HCO3-) Bicarbonate

(BSA) Bovine serum albumin

(Ca2+) Calcium

(Cl–) Chloride

(DNA) Deoxyribonucleic acid

(Dex) Dextranase

(H2PO4–) Dihydrogen phosphate

(ELISA) Enzyme linked immunosorbent assay

(FFPE) Formalin-fixed paraffine- embedded

(GCF) Gingival crevicular fluid

(GTF) Glucosyltransferase


IV

(HTN1) Histatin1

(HTN3) Histatin3

(HTLV-III) Human T-lymphotropic virus

( F–) Iron

(IgA) Immunoglobulin A

(I–) Iodine

(Mg2+) Magnesium

(miRNA) Micro-RNA

(PCR) Polymerase chain reaction

(K+) Potassium

(RSID) Rapid Stain Identification

(RT-PCR) Reverse transcription-polymerase chain reaction


V

(ROC) Receiver Operating Characteristic

(STR) Short tandem repeats

(STATH) Statherin

(Na+) Sodium

(SDS) Sodium dodecylsulfate

(qpcr) quantitative PCR

List of tablesList of tables

VI

No. Title Page1 The percentage of detection of S. salivarius from various body

fluid samples and skin swabs.

63

2 The percentage of detection of S. mutans from various body fluids

and skin swabs.

64

3 The percentage of detection of S.salivarius and s. mutans from all

saliva contained samples.

67

4 The percentage of detection of S. salivarius and S. mutans from

fresh saliva samples.

68

5 The percentage of detection of S. salivarius from saliva stain

(licked filter paper).

69


semen mixed with saliva samples.

71


cotton contaminated with saliva samples.

72


bitten apple contaminated with saliva samples.

74


cigarette butts samples.

75

List of tablesList of tables

VII

10 The percentage of detection of S. salivarius and S.mutans from

different forensic samples.

77

11 The significance between different forensic groups 79

12 Cross tabs between PCR method and API method in identification

of S. salivarius.

80

13 Cross tabs between PCR method and API method in identification

of S. mutans.

82

14 The ROC curve measuring the sensitivity and the specificity of

PCR and API methods for detection of S.salivarius.

85


PCR and API methods for detection of S.mutans.

88

List of figuresList of figures

VIII

No. Title Page1 Schematic of commonly used DNA extraction processes 34

2 DNA amplification process with PCR 42

3 Schematic of The 5 ′ Nuclease Assay (TAQMAN) 47

4 The identification of S. salivarius and S. mutans from various

body fluids and skin swabs.

65

5 An agarose gel electrophoresis show PCR products of

Streptococcus salivarius strain in different saliva contained

forensic samples.

66

6 An agarose gel electrophoresis show PCR products of

streptococcus mutans strain in different saliva contained

forensic samples.

66

7 The identification of S. salivarius and S. mutans identification

from fresh saliva samples.

68

8 The identification of S. salivarius and S. mutans from saliva

stain (licked filter paper) samples.

70

9 The identification of S. salivarius and S. mutans from semen

mixed with saliva samples.

71

10 The identification of s. salivarius and s. mutans from cotton

contaminated with saliva samples.

73

List of figuresList of figures

IX

11 The identification of S. salivarius and S. mutans from bitten

apple contaminated with saliva sample.

74

12 The identification of S. salivarius and S. mutans from cigarette

butts samples.

76

13 The identification of S. salivarius and S. mutans from different

forensic samples (Detected samples).

78

14 Cross tabs between PCR method and API method in

identification of S. salivarius.

81

15 Cross tabs between PCR method and API method in

identification of S. mutans.

83


S. salivarius detection by PCR and API methods.

87

17T The ROC curve measuring the sensitivity and the specificity of

S.mutans detection by PCR and API methods.

89

AbstractAbstract

Background: Oral streptococci represent about 20 % of the total oral

bacteria, so if it is possible to detect the presence of oral-specific bacteria by PCR

from a forensic specimen, this could be used to verify the presence of saliva.

Aim of the work: detection of Streptococcus salivarius which is one of the most

common streptococci in oral bacteria and streptococcus mutans which is

common in cases of dental caries in various body fluids and to asses which one

of them is more reliable in saliva identification. Materials and methods: control

samples were taken from various body fluids (urine, semen, fresh saliva, saliva

stain) and skin swabs. Forensic samples included (cotton fabrics contaminated

with saliva, cigarette butts, bitten apple and semen mixed with saliva samples).

DNA extraction was done using DNeasy ® blood and tissue kit (qiagen). PCR

was done for DNA amplification using PCR master mix then gel electrophoresis

was done for samples qualification. Control bacteria were streptococcus

salivarius and streptococcus mutans. Results and conclusion: streptococcus

salivarius was detected in 83. 5% of saliva samples and S.mutans was detected in

67% of saliva samples. Both bacteria were not detected in other body fluids, so S.

salivarius is more reliable in saliva identification as well as differentiating it from

other body fluids. Recommendations: PCR is valuable in detection of saliva by

detecting s. salivarius.

Key words: Saliva, Identification, PCR, S.salivarius, S.mutans.

Introduction

1

Introduction

Recent developments in forensic practices have contributed to the

investigation of crimes, the discrimination of body fluids in forensic examination is

important to determine the events that took place at the crime scene , the detection

of saliva is particularly important for understanding the details of crime

(Nakanishi et al., 2009).

Saliva may be found on victims of several violent crimes. It had been shown

that saliva can potentially be recovered and typed from bite marks, cigarette butts,

postage stamps, envelopes and other objects. Stains of dried saliva are invisible,

making its recognition and collection difficult (Kanto et al., 2005).

In a bite mark, tooth in combination with other mouthparts cause a mark on

victims' skin or some object, which can be compared with the unique

characteristics of a suspected biter's dentition by several methodologies (Wright

and Dailey, 2001). Besides the physical evidence present in a bite mark, there is

biological evidence that can assist the investigation. During the biting process,

saliva is deposited on the skin or object surface in enough amount to allow typing

of the deoxyribonucleic acid (DNA). For this purpose, the bite mark area is

swabbed using the standard bite mark operating procedures, and DNA can be

extracted and analyzed (Sweet and pretty, 2001).

Oral streptococci are major constituents of human oral flora that are

commonly found in the oral cavity and upper respiratory tract, mutans group

streptococci have been implicated as prime causative organisms of human dental

caries (Hoshino et al., 2004). Streptococcus salivarius is one of the most common

streptococci in oral bacteria , Polymerase chain reaction (PCR) have recently been

Introduction

2

used to detect and identify oral bacteria such as streptococcus salivarius and

streptococcus mutans (Chen et al., 2007).

With respect to forensics, the detection of oral streptococci had only used to

verify bite marks. However these reports did not discuss the identification of saliva

presence, if it was possible to detect the presence of oral specific bacteria by PCR

from a forensic specimen, this could be used to verify the presence of saliva

(Nakanishi et al., 2009).

Aim of the work

Streptococci are the most abundant oral bacteria and represent about 20% of

the total oral bacteria. This study is to prove a link between the presence of oral

streptococci especially S.salivarius and S.mutans and saliva detection. Therefore

we will asses if the detection of S. salivarius and S. mutans by PCR is sufficient to

confirm saliva presence and differentiating it from other body fluids and skin

swabs in forensic samples.

Review of literature

3

Chapter 1

Saliva Composition and Functions

Saliva, the most available and non-invasive biofluid of the human

body, is derived from several types of salivary glands. Each type of

salivary gland secretes saliva with characteristic composition and properties.

The secretions from these different glands have been shown to differ

considerably, to be complex in composition and to be affected by different

forms of stimulation, time of day, diet, age, gender, a variety of disease states,

and several pharmacological agents (Greabu et al., 2009).

Physiology of Saliva

Whole saliva is a mixed fluid that is derived predominantly from 3 pairs

of major salivary glands: the parotid, the submandibular, and the sublingual

glands. Approximately 90% of total salivary volume results from the

activity of these 3 pairs of glands, with the bulk of the remainder from

minor salivary glands located at various oral mucosal sites. The whole

saliva also contains gingival crevicular fluid (GCF), mucosal transudations,

expectorated bronchial and nasal secretions, serum and blood derivatives

from oral wounds, bacteria, and bacterial products, viruses and fungi,

desquamated epithelial cells, other cellular components, and food debris

(Amerongen et al., 2004).


4

Saliva is stored in secretion granules in the acini of the salivary glands.

These granules are filled with water, in which electrolytes and proteins are

dissolved. Even not stimulated, salivary glands secrete a fluid, which is

produced via vesicles and not by exocytosis. For this process, the saturation of

the glands with blood is of outmost importance. It is an energy demanding

process for which adenosinetriphosphate (ATP) is needed, which is generated

by metabolizing intracellular glycogen. Besides this exocytotic process, there

is also a paracellular source of fluid, coming from the interstitium, which is

especially the case when salivation is stimulated (Aps et al., 2005).

Saliva Composition

Saliva is a clear, slightly acidic (pH 6 -7) liquid composed of inorganic

components and organic components. Inorganic components include water

which is a major content (approximately 99%), followed by ions Na+,Cl–

Ca2+,K+,HCO3,H2PO4–, F–, I–,Mg2+, thiocyanate. The ionic composition of

saliva is different from the plasma although derived from it. The hypotonicity

facilitates taste sensitivity and hydrates various organic compounds that form a

protective coating on the oral mucosa. Resultant bicarbonate serves as a

buffering agent and calcium and phosphate neutralize acids that would

otherwise compromise tooth mineral integrity (Dodds et al., 2005).

Organic components such as: urea, ammonia, uric acid, glucose,

cholesterol, fatty acids, mono–, di–, and triglycerides, phosphor and neutral


5

lipids, glycolipids, amino acids, steroid hormones and proteins that aid in the

protection of oral cavity tissues, including mucins, amylases, agglutinins,

glycoproteins, lysozymes, peroxidases, lactoferrin and secretory IgA. Non-

immune factors include lactoferrin, lysozyme, myeloperoxidase, histatins,

cystatins, mucin G1 and G2, and defensins. In addition, these macromolecules

form a viscoelastic mucosal coat and tooth enamel pellicle that aggregate and

cleanse bacteria and debris from the oral cavity. Saliva contains a variety of

antimicrobial constituents and growth factors (Greabu et al., 2009).

The human oral microbial biota represents a highly diverse biofilm.

Twenty-five species of oral streptococci inhabit the human oral cavity and

represent about 20 % of the total oral bacteria. Oral streptococci encompass

friends and foes bacteria. Each species has developed specific properties for

colonizing the different oral sites subjected to constantly changing conditions,

for competing against competitors, and for resisting external aggressions (host

immune system, physicochemical shocks, and mechanical frictions).

Imbalance in the indigenous microbial biota generates oral diseases, and under

proper conditions, commensal streptococci can switch to opportunistic

pathogens that initiate disease in and damage to the host (Nicolas et al., 2011).

The average total microscopic count is approximately 750 million oral

bacterial cells per milliliter of saliva. Of these, streptococci are the most

abundant. Specifically, Streptococcus salivarius is one of the most common

streptococci in oral bacteria; the group of "mutans streptococci" was described


6

as the most important bacteria related to the formation of dental caries.

Streptococcus mutans, although naturally present among the human oral

microbiota, is the microbial species most strongly associated with carious

lesions (Nakanishi et al., 2009).

Mutans group streptococci (Streptococcus mutans and Streptococcus

sobrinus), mitis group streptococci (Streptococcus mitis, Streptococcus

sanguinis, Streptococcus oralis, and Streptococcus gordonii), and salivarius

group streptococci (Streptococcus salivarius, etc.) are major constituents of

human oral flora that are commonly found in the oral cavity and upper

respiratory tract. Mutans group streptococci have been implicated as prime

causative organisms of human dental caries. Although the cariogenic potential

of mitis and salivarius group streptococci, which are predominant colonizers

of tooth surfaces, is low, their interactions may be important for the

establishment and maintenance of oral microflora and cariogenic plaque

(Hoshino et al., 2004).

Saliva is a complex and dynamic biological fluid which contains a

myriad of compounds. The biochemical physical and chemical properties of

salivary components and their interaction, accomplish the numerous functions

that saliva performs in the oral cavity. Nowadays many assays are available to

analyze various salivary parameters; however, standardization of collection

and storage methods is essential to obtain meaningful results (Schipper et al.,

2007).


7

Biochemical Aspects of Saliva

Saliva plays an important role in oral food processing as it prepares the

food for swallowing by moistening, lubrication and bolus formation. Different

authors attempted to correlate salivary lubrication to perception of food or

swallowing characteristics. Especially fat enhances lubrication and ease of

swallowing. In addition, friction was correlated with fat perception and

astringency of emulsions (Stokes et al., 2004).

Human saliva contains a large array of proteins and peptides that have

important biological functions. The functions of most of salivary proteins are

still poorly understood, although protein components in saliva have been

partially revealed by conventional biochemical strategies focused on

individual molecules or specific group of salivary proteins. A promising new

approach to study saliva is the global analysis of salivary proteins using

proteomic techniques. Such exploration of the salivary proteome will not only

improve our knowledge of oral physiology, but can also allow the

identification of novel proteins and the examination of changes in protein

levels under different physiological conditions or pathologic states (Hu et

al., 2007).

Saliva as a source of Human DNA

Saliva is potentially a very good source of human DNA, as the mean

number of epithelial cells per 1 mL of saliva is about 4.3 ×10ˆ5, whereas

the number of nucleated cells in 1 mL of whole blood is about 4.5–11 ×

10ˆ5. Moreover, the turnover of epithelial cells is quite extensive in the mouth,

as the surface layer of epithelial cells is replaced, on average, every 2.7 h,


8

suggesting that there is likely to be intact genomic DNA in saliva samples.

Indeed, recent studies have shown that human genomic DNA can be reliably

obtained from saliva (Quinque et al., 2006).

On average, the amount of human DNA (as measured by a TaqMan-

based assay) was about 11.4 g/mL saliva, which is more than that could be

obtained from other noninvasive samples such as cheek swabs. However, the

presence of large amounts of nonhuman DNA (up to 90% of the total extracted

DNA) in saliva samples does necessitate DNA quantitation methods that are

specific for human DNA. Thus, saliva can be considered a reliable source of

DNA for a wide variety of genetic studies (Ng et al., 2004).

Forensic Analysis of Saliva

The capacity of detecting human DNA in saliva has also been useful in

forensics. Saliva can be found in many areas inside a crime scene such as in

bite marks left in objects or victims of violent crimes, cigars, postage stamps,

envelopes, and other objects. It had been shown that saliva could be

potentially recovered in such cases. A study conducted by (Kanto et al., 2005)

obtained the saliva of volunteers from their skin for the extraction and

identification of DNA through PCR, for evaluation of its utilization and its

contribution to forensic dentistry. The results indicated that standardized

procedures used for collection and extraction of salivary DNA can be used

as a method to recover DNA in forensic cases, since there were enough

quantities for analysis. This would allow such tests to be incorporated into the

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