POLYMERASE CHAIN REACTION docx

POLYMERASE
CHAIN REACTION

Edited by Patricia Hernandez-Rodriguez
and Arlen Patricia Ramirez Gomez










Polymerase Chain Reaction
Edited by Patricia Hernandez-Rodriguez and Arlen Patricia Ramirez Gomez


Published by InTech
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Copyright © 2012 InTech
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First published May, 2012
Printed in Croatia

A free online edition of this book is available at www.intechopen.com
Additional hard copies can be obtained from

Polymerase Chain Reaction, Edited by Patricia Hernandez-Rodriguez
and Arlen Patricia Ramirez Gomez
p. cm.
ISBN 978-953-51-0612-8

Contents

Preface IX
Chapter 1 Application of PCR in Diagnosis
of Peste des Petits Ruminants Virus (PPRV) 1
Muhammad Abubakar, Farida Mehmood,
Aeman Jeelani and Muhammad Javed Arshed
Chapter 2 Application of PCR-Based Methods to Dairy Products and to
Non-Dairy Probiotic Products 11
Christophe Monnet and Bojana Bogovič Matijašić
Chapter 3 Role of Polymerase Chain Reaction
in Forensic Entomology 51
Tock Hing Chua and Y. V. Chong
Chapter 4 PCR for Screening Potential
Probiotic Lactobacilli for Piglets 65
Maurilia Rojas-Contreras, María Esther Macías-Rodríguez
and José Alfredo Guevara Franco
Chapter 5 Polymerase Chain Reaction for Phytoplasmas Detection 91
Duška Delić
Chapter 6 Molecular Diagnostics of Mycoplasmas:
Perspectives from the Microbiology Standpoint 119
Saúl Flores-Medina, Diana Mercedes Soriano-Becerril
and Francisco Javier Díaz-García
Chapter 7 BRAF V600E Mutation Detection Using
High Resolution Probe Melting Analysis 143
Jennifer E. Hardingham, Ann Chua, Joseph W. Wrin,

Aravind Shivasami, Irene Kanter,
Niall C. Tebbutt and Timothy J. Price
Chapter 8 Polymerase Chain Reaction:
Types, Utilities and Limitations 157
Patricia Hernández-Rodríguez and Arlen Gomez Ramirez
VI Contents

Chapter 9 The Application of PCR-Based Methods
in Food Control Agencies – A Review 173
Azuka Iwobi, Ingrid Huber and Ulrich Busch
Chapter 10 PCR in Food Analysis 195
Anja Klančnik, Minka Kovač, Nataša Toplak,
Saša Piskernik

and Barbara Jeršek
Chapter 11 PCR in Disease Diagnosis of WND 221
Asifa Majeed, Abdul Khaliq Naveed,
Natasha Rehman and Suhail Razak
Chapter 12 Real-Time PCR for Gene Expression Analysis 229
Akin Yilmaz, Hacer Ilke Onen, Ebru Alp and Sevda Menevse
Chapter 13 Recent Advances and
Applications of Transgenic Animal Technology 255
Xiangyang Miao
Chapter 14 Measuring of DNA Damage by Quantitative PCR 283
Ayse Gul Mutlu
Chapter 15 Detection of Apple Chlorotic Leaf Spot Virus
in Tissues of Pear Using In Situ RT-PCR
and Primed In Situ Labeling 295
Na Liu, Jianxin Niu and Ying Zhao
Chapter 16 Application of PCR Technologies

to Humans, Animals, Plants and Pathogens
from Central Africa 309
Ouwe Missi Oukem-Boyer Odile, Migot-Nabias Florence,
Born Céline, Aubouy Agnès and Nkenfou Céline
Chapter 17 Study of Mycobacterium Tuberculosis
by Molecular Methods in Northeast Mexico 349
H. W.

Araujo-Torres, J. A. Narváez-Zapata,
M. G. Castillo-Álvarez, MS. Puga-Hernández,
J. Flores-Gracia and M. A. Reyes-López
Chapter 18 Development of a Molecular Platform
for GMO Detection in Food and Feed on the Basis of
“Combinatory qPCR” Technology 363
Sylvia Broeders, Nina Papazova,
Marc Van den Bulcke and Nancy Roosens
Chapter 19 Overview of Real-Time PCR Principles 405
Morteza Seifi, Asghar Ghasemi, Siamak Heidarzadeh,
Mahmood Khosravi, Atefeh Namipashaki, Vahid Mehri Soofiany,
Ali Alizadeh Khosroshahi and Nasim Danaei
Contents VII

Chapter 20 PCR Advances Towards the Identification of Individual and
Mixed Populations of Biotechnology Microbes 443
P. S. Shwed
Chapter 21 Lack of Evidence for Contribution of eNOS, ACE and AT1R
Gene Polymorphisms with Development of Ischemic Stroke
in Turkish Subjects in Trakya Region 455
Tammam Sipahi
Chapter 22 Analysis of Genomic Instability and Tumor-Specific Genetic

Alterations by Arbitrarily Primed PCR 469
Nikola Tanic, Jasna Bankovic and Nasta Tanic
Chapter 23 Analysis of Alternatively Spliced Domains
in Multimodular Gene Products - The Extracellular Matrix
Glycoprotein Tenascin C 487
Ursula Theocharidis and Andreas Faissner
Chapter 24 Submicroscopic Human Parasitic Infections 501
Fousseyni S. Touré Ndouo
Chapter 25 Identification of Genetic Markers Using Polymerase Chain
Reaction (PCR) in Graves’ Hyperthyroidism 517
P. Veeramuthumari and W. Isabel
Chapter 26 Detection of Bacterial Pathogens
in River Water Using Multiplex-PCR 531
C. N. Wose Kinge, M. Mbewe and N. P.

Sithebe
Chapter 27 PCR-RFLP and Real-Time PCR Techniques
in Molecular Cancer Investigations 555
Uzay Gormus, Nur Selvi and Ilhan Yaylim-Eraltan








Preface

This book is intended to present current concepts in molecular biology with the

emphasis on the application to animal, plant and human pathology, in various aspects
such as etiology, diagnosis, prognosis, treatment and prevention of diseases as well as
the use of these methodologies in understanding the pathophysiology of various
diseases that affect living beings.
It is known today that molecular biology has revolutionized the study and
understanding of health and disease. Significant developments occurred after 1953,
based on the impact generated in many disciplines, especially those life-related such
as medicine. Furthermore, the advances in molecular biology have revolutionized
industry, agriculture, pharmacology, and animal and plant production, among
others. Technology based on Molecular Chain reaction Polymerase (PCR) is
advancing rapidly since it is fundamental for improving the health of all living
beings.
Importantly, the most of the research in biology and medicine requires a series of
molecular strategies that allow the generation of new knowledge, in order to enable
better understanding of the mechanisms of life and the cellular changes that affect all
living things. Molecular biology has transformed the way we see and understand the
physiological and pathological changes of cells, organs and systems. In this sense, this
book presents the fundamentals, applications, advantages and disadvantages of
various molecular techniques from the research process in biology, medicine,
agriculture and environment in basic and applied science. Each chapter explains
molecular techniques through various experiments offering new knowledge in
different disciplines with applications trying to ultimately improve the conditions of
life.
The book includes the participation of different authors and co-authors of various
nationalities, all of them experts in the field. The book will be useful to professionals,
students, teachers and researchers interested in expanding their knowledge in
molecular biology, one of the most exciting areas of work today.
X Preface

I am grateful for the possibility of editing this book and sending a message to all

readers: perform with passion, responsibility and dedication your projects in life; in
my case – it is the research.

Patricia Hernandez-Rodriguez
Universidad De La Salle, Bogota,
Colombia




1
Application of PCR in Diagnosis
of Peste des Petits Ruminants Virus (PPRV)
Muhammad Abubakar
*
, Farida Mehmood,
Aeman Jeelani and Muhammad Javed Arshed
National Veterinary Laboratory (NVL), Park Road, Islamabad
Pakistan
1. Introduction
a. Global perspective of PPRV
A Peste des petits ruminant (PPR) is a viral disease of sheep, goats and wild ruminants. It is
acute disease which is endemic in many countries of Africa, Arabian Peninsula, Middle east
and India.
7, 12, 13

It was first reported in Côte d'Ivoire in West Africa
14
and was named as Kata,
psuedorinderpest, pneumoenterititis complex and stomatitis-pneumenteritis syndrome

15
.
Then in 1972 a sort of disease in goats in Sudan was identified to be PPR
16
. In recent years
either the presence of antibodies to the virus or viral nucleic acid has been confirmed from
the countries like Burkina Faso (2008), Ghana (2010), Nigeria (2007) and Senegal (2010)
17
.
Recently detection of PPRV in East Africa countries is shown by the detection of Antibodies
in Kenya (1999 and 2009) and Uganda (2005 and 2007)
18
. It has also been detected in North
Africa (Egypt) in 1987 and 1990.
In Saudi Arabia, an outbreak of PPRV has been reported in April, 2002 in Sheeps and Goats
1
.
In Pakistan PPRV has been reported since 1991 which was confirmed by PCR in 1994.
19
In
India the was first reported in 1987
11
. In Iran the disease was reported in 1995
20
while in
Iraq it was first detected in 2000
21
.
b. Disease picture of PPRV
Peste des petits ruminants (PPR) represents one of the most economically important animal

diseases in areas that rely on small ruminants. Outbreaks tend to be associated with contact
of immuno-naïve animals with animals from endemic areas. In addition to occurring in
extensive-migratory populations, PPR can occur in village and urban settings though the
number of animals is usually too small to maintain the virus in these situations.
• Morbidity rate in susceptible populations can reach 90–100%
• Mortality rates vary among susceptible animals but can reach 50–100% in more severe
instances

*
Corresponding Author

Polymerase Chain Reaction

2
• Both morbidity and mortality rates are lower in endemic areas and in adult animals
when compared to young ones.

Fig. 1. Geographic distribution of PPRV lineages (Dhar et al., 2002)
c. Hosts Range
• Goats (predominantly) and sheep
• Breed-linked predisposition in goats

Fig. 2. Clinical Picture and Severity of the Disease
Wildlife host range not fully understood
• documented disease in captive wild ungulates: Dorcas gazelle (Gazelle dorcas),
Thomson's gazelles (Gazella thomsoni), Nubian ibex (Capra ibex nubiana), Laristan
sheep (Ovis gmelini laristanica) and gemsbok (Oryx gazella)
• Experimentally the American white-tailed deer (Odocoileus virginianus) is fully
susceptible
Lineage 1

Lineage 2
Lineage 3
Lineage 4
Uncharacterised

Application of PCR in Diagnosis of Peste des Petits Ruminants Virus (PPRV)

3
• Cattle and pigs develop in-apparent infections and do not transmit disease
• May be associated with limited disease events in camels
2. Molecular epidemiology of PPRV
A close contact between the infected animals which is in the febrile stage and susceptible
animals is a source of transmission of the disease
15
. During sneezing and coughing the virus
spread from animal to animal
22
. Indirect transmission seems to be unlikely in view of the
low resistance of the virus in the environment and its sensitivity to lipid solvent.
4

Epidemiology pattern vary from area to area, for example in the humid Guinean zone where
PPR occurs in an epizootic form can cause mortality between 50-80% while in arid and semi-
arid regions, PPR is seldomly fatal but usually occurs as a subclinical or inapparent infection
opening the door for other infections such as Pasteurellosis
4
. In Saudi Arabia a high
morbidity of 90% was reported,
2
3-8 months animal are more susceptible to disease than

either of adults or unweaned animals
23
.
a. Genome Organization of PPRV:
PPRV belong to Morbillivirus genus. For a long time it was thought to be a variant of RP that
was adapted to sheeps and goats and had lost its virulence for cattles.
3
The causative agent
of PPR is RNA virus which is single strand and non-segmented. It belongs to the family
Paramyxoviridae and genus Morbillivirus which also includes measles virus, rinderpest virus
(RPV), canine-distemper virus, phocinedistemper virus, and dolphin and porpoise
morbilliviruses
24
. All the viruses belonging to the genus morbilli are serologically related.
Phylogenetic analysis also shows that there is high degree of homology.
The genome contains six tandemly arranged transcription units which encodes six structural
proteins i.e the surface glycoproteins F and H, the nucleocapsid (N), the matrix (M), the
polymerase or large (L) and the polymerase-associated (P) proteins. The cistron directing the
synthesis of this later protein is encoding the virus non-structural proteins C and V by the
use of two other open reading frames (ORF) of the messengers. The gene order is 3’N-P-M-
F-H-L5’, as determined by transcriptional mapping.
25
The genome is flanked by extragenic
sequences at the 3’ ((52 nucleotides, leader) and 5’ ends (37 nucleotides, trailer).
For viruses of the family Paramyxoviridae, the genome promoter (GP) contains 107
nucleotides comprising the leader sequence and the adjacent non-coding region of the N
gene at the 3’ end of the negative-strand. While antigenome promoter (AGP) contain 109
nucleotides that encompass the trailer sequence and the proximal untranslated region of the
L gene. Both GP and the AGP contains the polymerase binding sites and the RNA
encapsidation signals for the replication of the full genome while the production of

messengers (m-RNA) is a function of the GP
26
. So GP and AGP have an impact on the
virulence of virus.
Genes and promoters of Morbillivirus; the protein coding regions (N, P, V, C, M, F, H, and L),
noncoding intergenic regions and the leader and trailer regions along with the specialized
sequence motifs are shown. The genome promoter includes the leader sequence and the non
coding regions N at the 3' end of the genomic RNA. The antigenome promoter includes the
trailer sequence and the untranslated regions of the L gene at 5’ end. Gene start (GS) and
gene end (GE), enclosing the intergenic trinucleotide motifs are also shown.

Polymerase Chain Reaction

4


Fig. 3. Genome of PPR virus
b. Antigenic and Immunogenic Epitopes:
Surface glycoproteins hemagglutinin (H) and fusion protein (F) of morbilliviruses are highly
immmunogenic and helps in providing the immunity. PPRV is closely related to rinderpest
virus (RPV). Antibodies against PPRV are both cross neutralizing and Cross protective. A
vaccinia virus double recombinant expressing H and F glycoproteins of RPV has been
shown to protect goats against PPR disease though the animals developed virus-
neutralizing antibodies only against the RPV and not against PPRV. Capripox recombinants
expressing the H protein or the F protein of RPV or the F protein of PPRV conferred
protection against PPR disease in goats, but without production of PPRV-neutralizing
antibodies
27
or PPRV antibodies detectable by ELISA (Berhe et al, 2003). These results
suggested that cell-mediated immune responses could play a crucial role in protection. Goats

immunized with a recombinant baculovirus expressing the H glycoprotein generated both
humoral and cell-mediated immune responses.
28
The responses generated against PPRV-H
protein in the experimental goats are also RPV crossreactive suggesting that the H protein
presented by the baculovirus recombinant ‘resembles’ the native protein present on PPRV.
28

Lymphoproliferative responses were demonstrated in these animals against PPRV-H and
RPV-H antigens
28
. N-terminal T cell determinant and a C-terminal domain harboring
potential T cell determinant(s) in goats were mapped. Though the sub-set of T cells (CD4+
and CD8+ T cells) in PBMC that responded to the recombinant protein fragments and the
synthetic peptide could not be determined, this could potentially be a CD4+ helper T cell
epitope, which has been shown to harbor an immunodominant H restricted epitope in

Application of PCR in Diagnosis of Peste des Petits Ruminants Virus (PPRV)

5
mice
28
.Identification of B- and T-cell epitopes on the protective antigens of PPRV would
open up avenues to design novel epitope based vaccines against PPR.
Sheep and goats are unlikely to be infected more than once in their economic life
12
. Lambs or
kids receiving colostrum from previously exposed or vaccinated with RP tissue culture
vaccine were found to acquire a high level of maternal antibodies that persist for 3-4
months. The maternal antibodies were detectable up to 4 months using virus neutralization

test compared to 3 month with competitive ELISA
29
. Measles vaccine did not protect against
PPR, but a degree of cross protection existed between PPR and canine distemper.
30

3. Specimen collection, processing and shipment
Before collecting or sending any samples from animals with a suspected foreign animal
disease, the proper authorities should be contacted. Samples should only be sent under
secure conditions and to authorized laboratories to prevent the spread of the disease. In live
animals, swabs of ocular and nasal discharges, and debris from oral lesions should be
collected; a spatula can be rubbed across the gum and inside the lips to collect samples from
oral lesions. Whole, unclotted blood (in heparin or EDTA) should be taken for virus
isolation and PCR. Biopsy samples of lymph nodes or spleen may also be useful. Samples
for virus isolation should be collected during the acute stage of the disease, when clinical
signs are present; whenever possible, these samples should be taken from animals with high
fever and before the onset of diarrhea. At necropsy, samples can be collected from lymph
nodes (particularly the mesenteric and mediastinal nodes), lungs, spleen, tonsils and
affected sections of the intestinal tract (e.g. ileum and large intestine). These samples should
be taken from euthanized or freshly dead animals. Samples for virus isolation should be
transported chilled on ice. Similar samples should be collected in formalin for
histopathology. Whenever possible, paired sera should be taken rather than single samples.
However, in countries that are PPR-free, a single serum sample (taken at least a week after
the onset of clinical signs) may be diagnostic.
4. Laboratory diagnosis of PPR
a. Conventional Methods of PPRV Diagnosis
Conventional techniques such as the Agar Gel Immuno Diffusion (AGID) test are not
routinely used for standard diagnosis as they lack sensitivity when compared to other
assays. However, Haemagglutination tests (HA) and Haemagglutination Inhibition tests
(HI) tests can be used for routine screening purposes in control programmes as they display

comparative sensitivity alongside being simple to perform and cheap to produce.
Virus isolation in cell culture can be attempted with several different cell lines where
samples permit. Although Vero cells have been the choice for isolation and propagation of
PPRV, it is reported that B95a, an adherent cell line derived from Epstein-Barr virus-
transformed marmoset B-lymphoblastoid cells, is more sensitive and support better growth
of PPRV lineage IV as compared to Vero cells. More recently, Vero cells expressing the
SLAM receptor have been used as an effective alternative for isolation in cell culture. The
fragility of morbillivirus virions generally renders techniques such as virus isolation
redundant for routine diagnostic use, especially where sample quality is poor. Such

Polymerase Chain Reaction

6
techniques are also considered to be time-consuming and cumbersome. Virus isolation does,
however, play an important role from a research perspective.
ELISA tests using monoclonal antibodies are often used for serological diagnosis and
antigen detection for diagnostic and screening purposes. For PPR antibodies detection, the
competitive ELISA is the most suitable choice as it is sensitive, specific, reliable, and has a
high diagnostic specificity (99.8%) and sensitivity (90.5%). Immunocapture ELISA (ICE) is a
rapid, sensitive and virus specific test for PPRV antigen detection and it can differentiate
between RPV and PPRV and has been reported to be more sensitive than the AGID test.
For rapid diagnosis to enable a swift implementation of control measures, further
development and validation of pen-side tests such as the chromatographic strip test and the
dot ELISA that can be performed without the need for equipments or technical expertise are
highly desirable.

Sr
#
Test Name Acronym Application
(Lab or Field)

Feature Detected
(Antigen or
Antibody)
1 Agar gel immuno-diffusion AGID Both Both
2 Counter Immuno-
electrophoresis
CIEP Both Both
3 Dot enzyme immunoassay Lab Antigen
4 Differential immuno-histo-
chemical staining of tissue
sections
IH
staining

Lab

Antigen
5 Haemagglutination and
Haemagglutination
inhibition
tests
HA and
HI

Both Both
6 Immuno-filtration IF Lab Antigen
7 Latex agglutination tests LA Field Antigen
8 Virus isolation VI Lab Antigen
9 Competitive enzyme-linked
Immuno-sorbent assay

(c-ELISA)
cELISA Lab

Antibody

10 Novel sandwich ELISA sELISA Lab Antigen
11 Immuno-capture enzyme-
linked immunosorbent
assay
Ic-ELISA Lab Antigen
Table 1. Detail of conventional methods for the detection and confirmation of PPR

Application of PCR in Diagnosis of Peste des Petits Ruminants Virus (PPRV)

7
b. Molecular Methods for PPRV Diagnosis
Molecular techniques such as reverse transcription polymerase chain reaction (RT- PCR)
and nucleic acid hybridization are generally used. These genome based techniques are
largely used because of their high specificity and sensitivity. However, modern one step
real-time RT-PCR assays specific for PPRV and loop-mediated isothermal amplification
techniques are more sensitive techniques for PPRV detection but do not allow genetic typing
of positive samples. RT-PCR coupled with ELISA have also been used to increase the
analytical sensitivity of visualization of RT-PCR products and to overcome the drawbacks of
electrophoresis-based detection such as use of ethidium bromide, exposure to UV light etc.
The assay is reported to detect viral RNA in infected tissue culture fluid with a virus titre as
low as 0.01 TCID50/100 µL and has been reported as being 100 and 10,000 times more
sensitive than the sandwich ELISA and RT-PCR, respectively.
31

5. Potential and application of PCR technique for future advances in

diagnosis of PPR
Among the various techniques developed for the detection of PPRV, PCR technique has
been the most popular and highly sensitive tool so far for diagnosis of PPR. The routine
serological techniques and virus isolation are normally used to diagnose morbillivirus
infection in samples submitted for laboratory diagnosis. However, such techniques are
not suitable for use on decomposed tissue samples, the polymerase chain reaction (PCR),
has proved invaluable for analysis of such poorly preserved field samples. The PCR test
consists of repetitive cycles of DNA denaturation, primer annealing and extension by a
DNA polymerase effectively doubling the target with each cycle leading, theoretically, to
an exponential rise in DNA product. There placement of the polymerase now fragment by
thermo-stable polymerase derived from Thermus aquaticus (Taq) has greatly improved
the usefulness of PCR. These qualities have made the PCR one of the essential techniques
in molecular biology today and it is starting to have a wide use in laboratory disease
diagnosis. Since the genome of all Morbilliviruses consists of a single strand of RNA, it
must be first copied into DNA, using reverse transcriptase, in a two-step reaction known
as reverse transcription polymerase chain reaction (RT-PCR).Among the various
techniques developed for the detection of PPRV, however, polymerase chain reaction
(PCR) technique developed using F-gene primers has been the most popular tool so far,
for diagnosis as well as molecular epidemiological studies. RT-PCR using phospho-
protein (P) universal primer and fusion (F) protein gene specific primer sets to detect and
differentiate between PPR and RP are described by
8, 24, 32
developed a RT-PCR test, using
phosphoprotein (P) gene and fusion protein(F) gene specific primer sets to detect and
differentiate RPV and PPRV. They observed that RT-PCR was able to detect virus
secretion in ocular swabs at four days post infection (PI) in experimentally infected goats,
as compared to eight days PI by IcELISA. RT-PCR assay preclude the need for virus
isolation and, because of the rapidity with which completely specific results could be
obtained, the assay appeared to be the test of choice for PPRV detection. Relative
specificity and sensitivity of F-gene based RT-PCR with sandwich-ELISA was 100 and 12.5

percent, respectively
31
.

Polymerase Chain Reaction

8
6. Conclusion
The conventional techniques are largely replaced by genome-based detection techniques for
the diagnosis and confirmation of PPR virus. Molecular-biological techniques such as RT-
PCR and nucleic acid hybridization are now in use. These genome based techniques are
largely used because of their high specificity and sensitivity. However one step real-time
RT-PCR assays specific for PPRV and loop-mediated isothermal amplification techniques
are more sensitive techniques for PPRV detection.
7. References
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Bishr,B. (2004). Emergence of peste des petits ruminants virus in sheep and goats in
Eastern Saudi Arabia. Rev Elev Med Vet Pays Trop 57, 31–34.

[2] Abu Elzein, E.M.E., Hassanien, M.M., Alfaleg, A.I.A, Abd Elhadi, M.A., Housawi, F.M.T.
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Bishr,B. (2004). Emergence of peste des petits ruminants virus in sheep and goats in
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with attenuated RP virus. Res. Vet. Sci. 27: 321-324.
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animals. Rev. Sci. Tech. Off. Int. Epiz., 12: 865–72
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Maiduguri, Nigeria.
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modulates replication efficiency. Virus Research 100: 199-211.
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2
Application of PCR-Based Methods to Dairy
Products and to Non-Dairy Probiotic Products
Christophe Monnet
1
and Bojana Bogovič Matijašić
2

1
UMR782 Génie et Microbiol. des Procédés Alimentaires INRA, AgroParisTech,

Thiverval-Grignon
2
Institute of Dairy Science and Probiotics, Biotechnical Faculty, University of Ljubljana
1
France
2
Slovenia
1. Introduction
Many types of cheeses and fermented dairy products are produced throughout the world.
They contain various types of bacteria and fungi. In many cases, their exact microbiological
composition is not well known because the deliberately added microorganisms are only part
of the final microbiota. These microorganisms contribute to the manufacturing of the
product (aroma compound production, acidification, impact on texture, colour etc.).
Occasionally, dairy products may also be contaminated by spoilage microorganisms and
pathogens. PCR-based methods have many interesting applications for dairy products. They
can be used to detect, identify and quantify either unwanted or beneficial microorganisms.
They can also provide culture-independent microbial fingerprints. Another application is
the detection or the quantification of specific genes or groups of genes, such as those
involved in the generation of the functional properties. In addition, the abundance of
specific mRNA transcripts can be quantified by reverse transcription real-time PCR, which
is very useful for a better understanding of the physiology and activity of the
microorganisms present in dairy products.
Probiotics have been defined as ‘‘live microorganisms that, when administered in adequate
amounts, confer a health benefit on the host’’ (FAO/WHO, 2002). The deficiencies of the
quality of probiotic products in terms of too-low numbers or the absence of labelled species
are commonly observed. The facts that probiotic functionality is a strain specific trait and
that several probiotic strains have very similar phenotypic properties dictate the need for
more powerful and rapid methods than conventional cultivation-based methods which have
several disadvantages and very limited selectivity. The use of PCR based methods especially
has greatly expanded during recent years.

Conventional PCR, combined with gel electrophoresis, has been successfully used for the
genus-, species- or strain-specific determination of the presence of probiotic organisms in
the products or in the biological samples (faeces). An important feature of probiotics,
however, is the viability which is a prerequisite for the probiotic functionality. In this
regard, a common DNA-based quantification by real-time PCR is not very useful for
quantification purposes since the DNA released from dead or damaged cells also

Polymerase Chain Reaction

12
contributes to the results of analysis. One of the alternative approaches for selective
detection of viable bacteria is the treatment of the samples with DNA-intercalating dyes
such as ethidium monoazide (EMA) or propidium monoazide (PMA) that they can
penetrate only into membrane-compromised bacterial cells or dead cells where they are by
photo-activation covalently linked to DNA and prevent it from PCR amplification.
2. Application of PCR-based methods to dairy products
2.1 Nucleic acid extraction from dairy products
2.1.1 DNA extraction
Most of the DNA present in cheeses and other fermented dairy products is from the
microorganisms that are present. This DNA has to be purified before performing PCR
analyses. Dairy products are compositionally complex and there are several reports of dairy
matrix-associated PCR inhibition (Niederhauser et al., 1992; Rossen et al., 1992; Herman and
Deridder, 1993). One can distinguish two types of DNA extraction methods from dairy
products: either direct extractions, or extractions after prior separation of the cells from the
food matrix. In all cases, the DNA extraction protocols have to be adapted to the cheese
under investigation.
Most methods described in the literature involve prior separation of the cells (Allmann et al.,
1995; Herman et al., 1997; Serpe et al., 1999; Torriani et al., 1999; McKillip et al., 2000;
Coppola et al., 2001; Ogier et al., 2002; Randazzo et al., 2002; Ercolini et al., 2003; Furet et al.,
2004; Ogier et al., 2004; Baruzzi et al., 2005; Rudi et al., 2005; Rademaker et al., 2006; El-

Baradei et al., 2007; Lopez-Enriquez et al., 2007; Parayre et al., 2007; Rossmanith et al., 2007;
Trmcic et al., 2008; Van Hoorde et al., 2008; Alegría et al., 2009; Dolci et al., 2009; Zago et al.,
2009; Le Dréan et al., 2010; Mounier et al., 2010). The recovery of cells from milks or
fermented milks is easier to perform than from cheeses. In most cases, homogenisation of
the samples and casein solubilisation is done in a sodium citrate solution, using a
mechanical blender or glass beads, and the cells are recovered subsequently by
centrifugation. Part of the fat is eliminated at this step because it forms a layer at the surface
after centrifugation. Serpe et al. (1999) homogenised cheese samples in a Tris-HCl buffer
containing the non-anionic detergent Tween 20 to emulsify the fat fraction of the sample.
Depending on the type of cheese and the ripening stage, the cell pellet obtained after
centrifugation may contain a large amount of caseins. These may be removed by washing
the cell pellet with a buffer once or several times, and compounds such as Triton X-100 may
be added for a better removal (Baruzzi et al., 2005). Caseins may also be eliminated by
pronase digestion before recovery of the cells by centrifugation (Allmann et al., 1995; Furet
et al., 2004; Ogier et al., 2004; Flórez and Mayo, 2006). It has been reported that the recovery
of the bacterial cells may be improved by addition of polyethylene glycol during the
homogenisation step (Stevens and Jaykus, 2004). A matrix lysis buffer containing urea and
SDS combined with an homogenisation in a Stomacher laboratory blender has been used by
Rossmanith et al. (2007) to recover Gram-positive cells from various food samples, including
cheeses. In the procedure described by Herman et al. (1997) and Bonetta et al. (2008),
bacterial cells are recovered from homogenised cheese by centrifugation after chemical
extraction of fat and proteins. At the surface of some cheeses, for example smear-ripened
cheeses, there is a high microbial density, and therefore, a simple surface scraping is
sometimes sufficient to recover the microbial cells without need to eliminate the

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

13
components from the cheese matrix (Rademaker et al., 2005). After their recovery, the cells
are disrupted and DNA is purified from the lysed cells. Cell disruption may involve bead-

beating, addition of lytic enzymes such as lysozyme, lyticase, mutanolysin or lysostaphine,
addition chemical compounds, or a combination of these treatments. After cell lysis,
purification of DNA may be performed by classical phenol/chloroform extraction. Phenol is
a strong denaturant of proteins that leads to the partition of the proteins into the organic
phase and at the interface of the organic and aqueous phases. Procedures avoiding the use
of phenol, which is a toxic chemical, have been described. For example, Coppola et al.
(2001), Rademaker et al. (2006), and Moschetti et al. (2001) used a commercial kit containing
a synthetic resin which removes the cell lysis products that interfere with the PCR
amplification. Baruzzi et al. (2005), Trmcic et al. (2008), and Furet et al. (2004) used a
commercial kit in which proteins are eliminated by the use of a protein precipitation
solution. Column-based or DNA-binding matrix purification methods have also been used
(Rudi et al., 2005; Parayre et al., 2007; Zago et al., 2009; Le Dréan et al., 2010), sometimes as a
final purification step after phenol/chloroform extraction (Stevens and Jaykus, 2004; Lopez-
Enriquez et al., 2007). Separation of cells from the food matrix simplifies the subsequent
steps of DNA extraction because most undesirable compounds such as matrix-associated
reaction inhibitors are eliminated at the first step of extraction. In addition, large amounts of
cheeses (for example more than 10 grams) can be processed in each extraction, which yields
a large final amount of DNA. This is important in dairy products containing a low
concentration of cells, for example at the initial steps of cheese-manufacturing, where direct
DNA extraction is in most cases not possible. Furthermore, the separation of cells from the
dairy food matrix eliminates in some cases the need for cultural enrichment prior to
detection of pathogens. In contrast to RNA, it is unlikely that there is a large quantitative or
qualitative change of the DNA present inside of the cells during the separation of the cells
from the dairy food matrix. One of the drawbacks of the DNA extraction methods based on
cell separation is that some DNA may be lost during the separation, due to cell lysis,
especially for yeasts and Gram-negative strains.
In direct DNA extraction procedures (McKillip et al., 2000; Duthoit et al., 2003; Feurer et al.,
2004a; Feurer et al., 2004b; Callon et al., 2006; Monnet et al., 2006; Delbes et al., 2007; Masoud
et al., 2011), the cheese samples are first homogenised in a liquid solution by a method
involving bead-beating, a mortar and pestle or other mechanical treatments. Efficient

treatments of casein degradation and cell lysis, followed by phenol/chloroform extractions,
are then needed to remove most contaminating compounds. Contaminating RNA can be
removed by a treatment with RNase. Subsequent alcohol precipitation or column-based
purification is then used to further purify and/to concentrate the DNA. Carraro et al. (2011)
used a column-based purification method for direct extraction of DNA from cheese samples.
2.1.2 RNA extraction
Reverse transcription PCR analyses of RNA may be used in microbial diversity evaluation
or for the detection or quantification of mRNA transcripts. Like for DNA, there are two
types of extraction methods for RNA from dairy products, either direct extractions, or
extractions after prior separation of the cells from the food matrix. The amount of RNA that
can be recovered from dairy products is in general higher than for DNA. Indeed, the RNA
content of microbial cells is higher than DNA. For example, in Escherichia (E.) coli, Bremer
and Dennis (1996) reported a concentration varying from 7.6 to 18.3 µg of DNA per 10
9
cells,