Detection Final

8/2/2019 Detection Final

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An Assignment

On

Detection of pathogens using

Genetic Probes

Submitted to: Dr. Vijendra Mishra

Professor

Dairy Microbiology Dept.

Submitted by: Ms. Ripan P. Goswami

M.Sc. -Dairy Microbiology

Reg. No. 04-1143-2009



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Introduction

A pathogen, (from Greek (pathos) "suffering, passion", (gen) "I give birth to")

an infectious agent, or more commonly germ, is a biological agent that causesdisease to its host. Foodborne illness (also foodborne disease and colloquially referred

to as food poisoning) is any illness resulting from the consumption of contaminated

food. There are two types of food poisoning: infectious agent and toxic agent. Food

infection refers to the presence of bacteria or other microbes which infect the body

after consumption. Food intoxication refers to the ingestion of toxins contained

within the food, including bacterially produced exotoxins, which can happen even

when the microbe that produced the toxin is no longer present or able to cause

infection. In spite of the common term food poisoning, most cases are caused by a

variety of pathogenic bacteria, viruses, or parasites that contaminate food, rather

than chemical or natural toxins.

Foodborne pathogens continue to cause major public health problems

worldwide. These organisms are the leading causes of illness and death in less

developed countries, killing approximately 1.8 million people annually. In developedcountries Foodborne pathogens are responsible for millions of cases of infectious

gastrointestinal diseases each year, costing billions of dollars in medical care and lost

productivity. In addition, new Foodborne diseases are likely to emerge driven by

factors such as pathogen evolution, changes in agricultural and food manufacturing

practices, and changes to the human host status. A third problem is that there are

growing concerns that terrorists could use pathogens to contaminate food and water

supplies in attempts to incapacitate thousands of people and disrupt economic

growth. Fuelled by these concerns research into the genomics, molecular biology and

microbiology of the most important Food borne pathogens has escalated to

unprecedented levels in recent years.

Quality control (QC), which is a reactive system that focuses on legal

requirements and emphasizes statistically relevant measurements, quality assurance

(QA) is a preventive approach that emphasizes operational procedures. To establishQA/QC parameters, the food microbiologist uses two approaches. The first sets out to



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determine the total load of microbes in a sample, and the second attempts to

determine the presence or absence of a particular microbial species, usually a

pathogen or related type used as their indicators. Thus, while the first type of

microbiological quality assurance test aims to establish that food products meet

statutory requirements, the second type of analysis is focused on public health

impacts with regulatory requirements as an integral part of the testing procedure. In

addition to the general testing requirements under QC programs, there has been an

added element of quality assurance that is being pursued vigorously under the

implementation of quality management systems such as Hazard Analysis & Critical

Control Points (HACCP) plans. To further enhance the utility of these systems, there

is a need to develop rapid microbiological detection techniques that are sensitive and

accurate. Accordingly, much effort has been devoted to shortening assay times and

to replacing the visible end results with alternative measurements.

Historically, the identification of such organisms has been done using

conventional culture methods and biochemical techniques. These methods, although

still considered by most to be the "gold standard" for microbial identification, can

often be time consuming, laborious and also complicated by the heterogeneous

nature of food microflora and the subjective nature of many microbiological and

biochemical techniques. Several bio-techniques are these days employed by

microbiologists to assure the safety of food products. These include electrical

methods such as impedance/conductance; chemical methods such as direct

epifluorescent filter technique (DEFT); bacterial adenosine triphosphate (ATP)

bioluminescence, flow cytometry, biosensors, and agglutination/immunological

assays; and nucleic acid technologies such as polymerase chain reaction (PCR),

ribotyping and microarrays. Nucleic acid technologies are being used increasingly

for quality assurance purposes. Such new methods have helped improve the

objectivity of test methods as well as reduce the time required to test many food

products.

DNA probe based methods have become increasingly popular in recent years.

The specific characteristics of any organism depend on the particular sequence of



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the nucleic acid contained in its genome. The sequence of bases of nucleic acids

makes different organisms unique. Detection of such organism’s unique nucleic acid

sequence employing short specific nucleic acid fragments which are labelled helps

in indicating the presence or absence of that particular organism in the sample.

Fig. 1 Detection of nucleic acids by amplification

Genetic Probe

Genetic Probe is a fragment of DNA or RNA of variable length (usually 100-

1000 bases long), which is used in DNA or RNA samples to detect the presence of

such nucleotide sequences (the specific DNA target) that are complementary to the

sequence in the probe. A gene probe is composed of nucleic acid molecules, most

often double-stranded DNA. It consists of either an entire gene or a fragment of a

gene with a known function. Alternatively, short pieces of single-stranded DNA can

be synthesized, based on the nucleotide sequence of the known gene. These are

commonly referred to as oligonucleotides. Both natural and synthetic

oligonucleotides are used to detect complementary DNA or RNA targets in samples.

Double-stranded DNA probes must be denatured before the hybridization reaction;

oligonucleotide and RNA probes, which are single-stranded, do not need to bedenatured.

The physical basis for gene probe tests stems from the structure of DNA

molecules themselves. Usually, DNA is composed of two strands of nucleotide

polymers wound around each other to form a double helix. These long nucleotide

chains are held together by hydrogen bonds between specific pairs of nucleotides.

Adenine (A) in one strand binds to thymine (T) in the complementary strand.Similarly, guanine (G) in one strand forms a hydrogen bond with cytosine (C) in the



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opposite strand. The hydrogen bonds holding the strands together can usually be

broken by raising the pH above 12 or the temperature above 95°C. Single-stranded

molecules result and the DNA is considered denatured. When the pH or temperature

is lowered, the hydrogen bonds are re-established between the AT and GC pairs,

reforming double-stranded DNA. The source of the DNA strands is inconsequential

as long as the strands are complementary. If the strands of the double helix are from

different sources, the molecules are called hybrids and the process is termed

hybridization. The probe thereby hybridizes to single-stranded nucleic acid (DNA or

RNA) whose base sequence allows probe-target base pairing due to complementarity

between the probe and target. The labeled probe is first denatured (by heating or

under alkaline conditions such as exposure to sodium hydroxide) into single DNA

strands and then hybridized to the target DNA (Southern blotting) or RNA (Northern

blotting) immobilized on a membrane or in situ

To detect hybridization of the probe to its target sequence, the probe is tagged

(or labelled) with a molecular marker of either radioactive or (more recently)

fluorescent molecules; commonly used markers are

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P (a radioactive isotope of phosphorus incorporated into the phosphodiester bond in the probe DNA) or

Digoxigenin, (DIG labels) which is non-radioactive antibody-based marker. DNA

sequences or RNA transcripts that have moderate to high sequence similarity to the

probe are then detected by visualizing the hybridized probe via autoradiography or

other imaging techniques. Normally, either X-ray pictures are taken of the filter, or

the filter is placed under UV light. Detection of sequences with moderate or high

similarity depends on how stringent the hybridization conditions were applied —

high stringency, such as high hybridization temperature and low salt in

hybridization buffers, permits only hybridization between nucleic acid sequences

that are highly similar, whereas low stringency, such as lower temperature and high

salt, allows hybridization when the sequences are less similar. Hybridization probes

used in DNA microarrays refer to DNA covalently attached to an inert surface, such

as coated glass slides or gene chips, and to which a mobile cDNA target is

hybridized.

http://en.wikipedia.org/wiki/Base_pair

http://en.wikipedia.org/wiki/Denaturation_(biochemistry)

http://en.wikipedia.org/wiki/Alkaline

http://en.wikipedia.org/wiki/Fluorescent_in_situ_hybridization

http://en.wikipedia.org/wiki/Isotopes_of_phosphorus


http://en.wikipedia.org/wiki/Autoradiography

http://en.wikipedia.org/wiki/Autoradiography


http://en.wikipedia.org/wiki/Fluorescent_in_situ_hybridization

http://en.wikipedia.org/wiki/Alkaline

http://en.wikipedia.org/wiki/Denaturation_(biochemistry)

http://en.wikipedia.org/wiki/Base_pair



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Depending on the method the probe may be synthesized using

phosphoramidite method or generated and labeled by PCR amplification or cloning

(older methods). In order to increase the in vivo stability of the probe RNA is not

used, instead RNA analogues may be used, in particular morpholino. Molecular

DNA- or RNA-based probes are now routinely used in screening gene libraries,

detecting nucleotide sequences with blotting methods, and in other gene

technologies like microarrays.

History

The first commercially available nucleic acid probe-based assay, GENE-TRAK,

was introduced in 1985 for food analysis. This used Salmonella -specific DNA probes

(32P labelled) directed against chromosomal DNA to detect Salmonella in enriched

foods. Later, in 1988, they introduced non-isotopically labelled probes

for Salmonella, Listeria and E. coli based on targeting the ribosomal RNA. This type of

colorimetric hybridization assay is based on a liquid hybridization reaction between

the target rRNA and two separate DNA oligonucleotide probes (the capture probe

and reporter probe) that are specific for the organism of interest. The capture probe

molecules are extended enzymatically with a polymer of about 100 deoxyadenosine

monophosphate residues. The reporter probe molecules are labeled chemically with

hapten fluorscein. The GENE-TRAK microwell tests available today are used to

detect Salmonella and Listeria . Other commercially available nucleic acid probes

exist for the confirmation of Campylobacter, Staphylococcus aureus and Listeria and

are marketed by Geneprobe. Initially, most such methods had limited utility owing to

the use of isotopic signal generating systems and complex assay formats. More

recently, however, a variety of format improvements and the development of more

sensitive nonradioactive detection methods have helped to broaden the application

of probe-based methods



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Target Selection

The first step in developing a gene probe assay is to decide what information is

needed. If a particular taxonomic group is to be identified, the probe must be

directed toward a gene or region of a gene that is conserved throughout a particular

species or genus. On the other hand, one may want to know if a microorganism

carrying a particular gene is present. Probes to specific determinants of virulence are

useful in assessing a risk to public health posed by bacterial contamination.

Table 1 lists probes that have been used or are of potential use for detecting bacterial

pathogens in foods. In the section, "Probes and Their Targets," the development of

each probe is described briefly along with what is known about the probe target andits significance. The first probes designed to detect all members of a taxonomic

group were constructed by screening randomly cloned DNA fragments. As data on

the evolution of ribosomal RNA nucleotide sequences accumulate, probes are being

directed toward these targets. Conserved regions can be used to identify large taxons,

whereas the variable regions may be unique for a particular genus or species.

Furthermore, as a cell contains more than 1000 copies of ribosomal RNA, test

sensitivity is increased, because fewer cells are required to produce a positive signal.

Probe Specificity

The relatively short length of synthetic oligonucleotide probes means that they

are specific for particular regions of DNA. There is only about 1 chance in 15,000

that a sequence length of 18 bases would appear more than once in

the E. coli genome. With a 22-base probe, the chance drops to about 1 in 4 million.

To avoid mismatches that reduce specificity, filter washings are conducted at high

stringency so that a single base-pair difference between target and probe could not

result in hybridization and produce a negative result. Such changes occur as the

result of rare mutations. The use of two non-overlapping probes would significantly

reduce the probability of false negatives.



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Construction of Probes

Recombinant DNA techniques have made gene probes possible. Probe tests

require preparations of relatively pure, specific segments of DNA. The first probes

were obtained by inserting these regions into plasmids and transforming the

plasmids into the appropriate host cells to increase the amount of probe DNA.

Plasmids were purified, and in some cases the inserted fragments were isolated.

These cloned, natural DNA probes served quite well, although a considerable

amount of effort was required for their production and purification. Through the

development of DNA sequencing and automated oligonucleotide synthesis, short(18-30 bases) DNA probes were produced in the laboratory by chemical means. The

ready availability of probes considerably expanded their use and application.

Table 1. Some gene probes used to detect pathogenic bacteria in foods

Bacteria Target(a)

Campylobacter jejuni r-RNA

Escherichia coli Heat-stable toxin (ST)

Heat-labile toxin (LT)Shiga-like toxin

Invasive genes

O157: H7

Listeria species r- RNA

L. monocytogenes Listeriolysin O

Major secreted polypeptide (msp)

Salmonella species r-RNA

Shigella species Invasive genes

Staphylococcus aureus Enterotoxin B

Vibrio cholera Cholera toxin

V. parahymolyticus Thermostable direct haemolysin (tdh)

V. vulnificus Cytotoxin- hemolysin

Yersinia en terocolitica Cytotoxicity/ Sereny

Inavasive gene (ail)

Yersinia pseudotuberculosis Invasive gene



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Probe Labeling

For probes consisting of cloned DNA fragments, the nick translation method

for labeling DNA with radioactivity is very popular. Cloned DNA can also be labeled

by a random priming technique. Several kits to perform these reactions are

commercially available; however, these techniques are unsatisfactory for labeling

short oligonucleotides. Oligonucleotide probes are usually labeled on the 5' end

with 32P, using bacteriophage T4 polynucleotide kinase and gamma-AT 32P.

Although radioactive gene probes seem to have the greatest sensitivity in colony

hybridization procedures, they are a potential biohazard, and disposal of radioactive

waste can be expensive.Many schemes are being examined for the nonradioactive labeling of gene probes.

Some of these techniques have been incorporated into commercial tests designed to

signal the presence or absence of a particular gene. For example, alkaline

phosphatase has been conjugated to synthetic oligonucleotides without affecting the

kinetics or specificity of the hybridization reaction.

Colorimetric DNA probe hybridization (DNA Probe Test- Dip stick format)

The probes used in this test consist of a mixture of capture probes and

detector probes. The capture probes contain two specific binding regions. The first

region contains a Salmonella- specific nucleic acid sequence. The second region

contains a polydeoxyadenylate tail. This serves to link the probe target complex to a

polydeoxythymidylatecoated solid support. Similarly, the detector probes alsocontain Salmonella-specific sequences. These sequences are labeled with fluorescein

groups that serve to bind an antibody-enzyme conjugate to the immobilized probe-

target complex. After removal of all of the excess reactants and cellular debris, the

plastic dipstick containing the immobilized probe-target complex is added to a

substrate-chromogen mixture in order to generate a highly colored product when

Salmonella is present in the original test sample (Fig.1). Hence indicating the

presence or absence of pathogen in the sample.



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In recent years, several genetic amplification techniques have been developed,

with polymerase chain reaction and its variants — nested PCR, reverse transcriptase

(RT) PCR and multiplex PCR — emerging as a biotechnique of choice in the food

industry. PCR involves detection of specific gene fragments by in-vitro enzymatic

amplification of the target DNA, followed by detection of the amplified DNA

molecule by electrophoresis, ELISA or other techniques. The PCR method is a highly

specific and sensitive method allowing the detection of low numbers of

microorganisms. In the past, the general limitations of the technique have included

difficulty in obtaining specific DNA primers and production of nonspecific PCR

products. Also, organisms that have been killed during processing were not

recognized as dead if their DNA was still present, thus giving false-positive reactions.

Automation and improvements in PCR systems have addressed some of these

problems and made the technique an extremely attractive option. Currently, there

are several manufacturers producing PCR-based test methods for the detection of

foodborne pathogens, including the BAX System Assays for Screening (DuPont

Qualicon, USA) forSalmonella, Listeria

, andE.coli

O157; Roche DiagnosticsLightCycler (Roche Diagnostics, USA); Probelia (Sanofi-Pasteur, France)

for Salmonella and Listeria , Genevision for Listeria monocytogenes,

Salmonella and E. coli O157:H7 (Warnex, Canada), and TaqMan (Perkin Elmer,

USA) for Salmonella.



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Fig.2 A schematic representation of the steps required to detect Salmonella using the DNA probe test

Colony Hybridization

DNA hybridization tests may be performed in many ways. One format, the

colony hybridization assay, will be described here. Generally, an aliquot of a

homogenized food is spread-plated on an appropriate agar. After incubation, the



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colonial pattern is transferred to a solid support (usually a membrane or paper filter)

by pressing the support onto the agar surface. Next, the cells are lysed in situ by a

combination of high pH and temperature (0.5 M NaOH and/or steam or microwave

irradiation), which also denatures and affixes the DNA to the support. The solid

support with the attached target DNA is incubated with a 32P- or enzyme-labeled

probe. The labeled probe DNA that fails to reform the double helix is removed by

washing the probe-target complexes on the support at an appropriate temperature

and salt concentration.

Great care must be taken to ensure that the washing temperature is correct;

this parameter is usually determined empirically. If the temperature of the washingsolution is too high, all the hydrogen bonds between the probe and target may be

broken, producing a false-negative result. If the washing temperature is too low,

strands of DNA will not match up accurately, and non complementary strands may

be formed, leading to a false-positive outcome. If the temperature allows only

accurately rejoined strands to remain together, the conditions are termed "high

stringency." If the temperature is too low, so that mismatched strands exist, the

stringency is low. For a review of hybridization using solid supports, Meinkoth and

Wahl.

The radioactive probe DNA that is bound to the target on the support is often

detected by autoradiography. An X-ray film is placed over the support. Radioactive

decays expose the film, so that when it is developed, black spots appear where cells

are harboring the same gene as the probe (Fig. 2). If an enzyme-labeled probe is

used, a chromogenic substrate is added. Where the probe-associated enzyme ispresent, a colored spot will develop. Each spot represents a bacterial colony that has

arisen from a single cell. The number of cells harboring the target gene in the

original sample can be calculated by multiplying the number of spots by the dilution

factor.



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Fig. 3. Colony Hybridization Assay

Aliquot of homogenized sample is spread-plated on appropriate medium (cross-

hatched area) and incubated until colonies are formed. Colonies are transferred by

gentle contact to solid support such as a filter (hatched area). Colony cells are lysed

in situ by high pH and/or steam or microwave irradiation, which immobilizes

single-stranded target DNA. Filters are then incubated with a labeled gene probe. (In

this figure, a radioactive label was used.) Unbound probe is removed by washing thefilter at a temperature that allows well-matched double strands to remain joined;

poorly matched strands are separated. If DNA from a colony contains the same

genetic information as the probe, that area of the filter will become radioactive.

Radioactivity is observed as a dark spot on an X-ray film. Count the spots to calculate

the number of cells containing specific gene present in the original sample.

The Polymerase Chain Reaction

Nucleic acid amplification techniques have an enormous range of

applications and have become an indispensible tool in molecular biology and

powerful rapid screening method in the detection of food borne pathogens. By

targeting and amplifying (or making copies of) DNA sequences in vitro , it has been

possible to detect the presence of specific DNA sequences with sensitivities down to a

single target copy per reaction, and in many cases quantify the results. PCR is a



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method for the amplification of double or single stranded (ss) DNA sequences in

vitro (Erlich, H.A et.al., 1989). The reaction proceeds in response to temperature

driven steps of double stranded (ds) DNA denaturation, primer or ss oligonucleotide

annealing to complementary ss target DNA sequences, and DNA polymerase

extension. These steps are repeated, and under appropriate conditions will generate

a doubling of the initial number of target copy sequences with each cycle. The

primers define the 5’ ends of the discrete products that are subsequently formed.

Three step PCRs use three individual temperature steps for denaturation, annealing,

and extension, while two step PCRs use a combined annealing and extension step.

Reaction reagents typically include a thermostable DNA polymerase,

deoxyribonucleoside triphosphates (dNTPs), user selected primers for targeting

specific sequences, magnesium chloride, and template or target DNA. The process is

rapid, requiring between minutes and hours to generate enough discrete sized target

sequences for detection; a single thermal cycle may require as little as a few seconds

to complete. The length of time required for a reaction is typically a function of

variables such as the length of the target sequence and the heating and cooling rates

of the thermal cycler used.

However, it is now possible to find PCR systems capable of thermal cycling

speeds so fast that decreasing cycle time further would not be worthwhile without

first finding a DNA polymerase capable of working faster than those currently in

use. While it is possible for PCR to routinely detect low copy numbers in a reaction,

many reactions use between 5 and 10 μl sample volumes, yielding a lower detection

limit of close to 103 CFU/ml. Among the expanding array of nucleic acid

amplification techniques, PCR remains the most popular method, presumably as a

result of its cost and ease of use and has been used extensively for the detection of

food borne pathogens. By the early 1990s numerous primer sets had been developed

for the detection of pathogens and the food industry had gained interest in this

powerful method. The amplification of nucleic acids for detection purposes is

usually just one step of a procedure that involves assay design and sample

preparation prior to amplification, followed by specificity and sensitivity analysis.



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Fig. 4 Representation of PCR and Detection Procedures

The advent of gene probe techniques has allowed the development of powerful

tests by which particular bacterial strains can be rapidly identified without the need

for isolating pure cultures (Rasmussen et al., 1994; Cohen et al, 1993). The

polymerase chain reaction (PCR) is a technique for in vitro amplification of specific

segments of DNA by using a pair of primers (Nguyen et al., 1994). A million-fold

amplication of a particular region can often be realized, allowing, among a myriad of

other uses, the sensitive detection of specific genes in samples. PCR can be used toamplify genes specific to taxonomic groups of bacteria and also to detect genes

involved in the virulence of food-borne bacteria (Finlay & Falkow, 1988; Bej et al,

1994). The recently developed techniques for amplifying specific DNA sequences in

vitro allow the detection of very small amounts of target DNA in various specimens.

Theoretically these procedures can detect even one molecule of target DNA. By

amplifying a sequence that is unique to the pathogenic micro-organism of interest,



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the in vitro amplification methods can be used to indirectly detect extremely low

concentrations of microbes.

However, provided that PCR products are handled carefully and that real-

time quantification is not necessary, traditional PCR techniques can be used with great

success for the detection of food pathogens. A single enrichment, thermal cycling

protocol, set of PCR reagent components and concentrations were used for the

detection of 13 foodborne pathogens by Wang et al. Agarose gel electrophoresis on

2% agarose gels stained with ethidium bromide was used for separation of PCR

products. The PCR detection limits reported ranged from 2 to 5 X 104 cells for E. coli

O157:H7 and Shigella spp., respectively. A review of PCR has been published. PCR hasbeen used to detect enteroinvasive E. coli and Shigella spp., V . Vulnificus , Hepatitis A

virus and V . cholerae in foods.

Food-borne pathogen identification is an important aspect of human health

care. PCR methods have been developed for the identification of Salmonella pathogens

(Hill, 1996; Jones et al., 1993; Tsen et al., 1994). PCR is an effective, rapid, reliable and

sensitive technique for the detection of fimA gene of Salmonella strains (Cohen et al.,

1993). Here, the primers selected were completely internal to the fimA gene, which

meant that all non-Salmonella strains responded negatively to the amplicon of the

fimA gene, making this a promising diagnostic tool for sensitivity and specificity.

Real Time PCR

The ability to monitor amplicon accumulation as a reaction proceeds has

drastically improved the field of nucleic acid detection. In addition to facilitating the

quantification of initial target copy numbers, real-time PCR allows an operator to

evaluate product specificity without opening the reaction chamber, saving time, and

reducing carry-over contamination risk. Real-time PCR systems offer a wide range of

capabilities. These include the ability to handle thousands of samples per day, perform

35 thermal cycles in under 40 minutes, and detect initial target copy numbers over a

range from 10 to 1010. The design of real-time PCR assays has been aided by



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commercially available software packages that can determine optimal primer, probe,

and reaction conditions, given a specific sequence of interest. Real-time PCR assays

are typically designed to target short DNA fragments using primers specifically

selected to avoid the formation of primer dimers. The increase in fluorescence in

response to amplicon formation is generally accomplished in one of two ways:

through the use of a nonspecific dsDNA binding, or by sequence specific probes that

generate a signal only in the presence of the target DNA sequence. Realtime PCR

techniques and applications have been reviewed extensively and experimental

comparisons among instrumentation and assay formats have been performed to

compare sensitivities. A large number of real-time PCR strategies that are based on

fluorescene increases in response to sequence specific detection have also been

developed. Probe based real-time PCR techniques are advantageous over the use of

nonspecific dsDNA binding dyes in that they may not require analysis of PCR

amplicon melting temperatures for product specificity — fluorescence generation is a

function of the probe binding to a specific sequence of DNA. In the case of real-time

PCR development with probe based systems, excitation and emission wavelengths of

the fluorophores selected must be kept in consideration.

Sequence specific chemistries that have been incorporated into real-time PCR

assays include those based on a sequence specific probe and DNA polymerase

exonuclease activity, molecular beacons, and self-quenched hairpin primers. One

real-time PCR chemistry (TaqMan®) that has been used extensively for the detection

of foodborne pathogens relies upon the 5’ exonuclease activity of Taq polymerase.

A probe containing a reporter and quencher in close proximity to one another

binds to a target region between the two primers which define the ends of the

discrete fragment ultimately formed. This probe is cleaved by the 5’ exonuclease

activity of a DNA polymerase, separating the fluorophores and quencher, generating

increases in fluorescence as a direct result of specific probe binding and target

fragment extension (Figure 1.2). Numerous assays have been developed with this

chemistry. Molecular beacons are stem and loop oligonucleotides structures used for



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sequence specific detection. The loop portion contains a sequence that is

complementary to a chosen target, while the stem portion contains a short sequence

of bases at the 3’ and 5’ ends that are complementary to one another but not the

target.(Fig. 4) Fluorescence and quenching moieties are attached to the ends of the

beacon. The beacons are designed such that with no loop complementary sequence

present the stem structure is stable, but in the presence of a complementary target

sequence the arms of the stem separate. This separation changes the conformation of

the beacon to a more stable structure, allowing simultaneous separation of the

fluorophore and quencher, leading to fluorescence generation. Molecular beacons

have been used in numerous applications, outside of monitoring specific amplicon

formation in real-time PCR. Molecular beacons have been used in multiplex PCR

applications for the simultaneous detection of four pathogenic retroviruses and

fourV. cholerae genes.

Reverse Transcriptase PCR

Enrichment procedures have successfully been used for the sensitive detection

of viable foodborne pathogens, but this technique is time consuming, as it is a

function of the target organisms growth. While PCR is capable of detecting low

levels of target

Fig. 4 (A) Molecular beacon stem-loop conformation that forms by intramolecular base pairing when in

solution without the presence of complementary target nucleic acid. (B) When in the presence of target DNA

or RNA, the molecular beacon unfolds because the bases comprising the loop (probe) region form more

numerous and more stable base pairs than those allowing the stem-loop secondary structure to form. A single

base mismatch between the target nucleic acid and the probe portion drastically decreases stability of

molecular beacon interaction and may preclude it altogether.



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DNA, DNA detection does not provide information regarding the viability of a cell;

food processing may destroy bacteria while leaving behind DNA and this DNA may

be present even if its host cell is no longer alive. On the other hand, RNA is easily

destroyed, which makes it suitable for determining organism viability. Reverse

transcriptase PCR of mRNA targets has demonstrated that these molecules are

indicators of cell viability. Following RNA purification and degradation of

contaminating DNA from a sample of interest, RNA is reverse transcribed and the

synthesized complementary DNA or cDNA may be amplified as is typically done for

any DNA target. Reverse transcriptase PCR has been used successfully for the

detection of foodborne bacterial pathogens55 and viruses. A real-time reverse

transcription PCR assay using a TaqMan minor grove binding probe was

implemented for the quantitative detection of H5 avian influenza down to 100 target

copies.

Microarray Detection

In the past several years, a new technology, called DNA microarray, has

attracted tremendous interests among biologists and offers much in the way of high

throughput analysis of virulence gene expression in food associated pathogenic

bacteria. This technology promises to monitor the whole genome on a single chip so

that researchers can have a better picture of the interactions among thousands of

genes simultaneously. An array is an orderly arrangement of samples. It provides a

medium for matching known and unknown DNA samples based on base-pairing

rules and automating the process of identifying the unknowns. An array experiment

can make use of common assay systems such as microplates or standard blotting

membranes, and can be created by hand or make use of robotics to deposit the

sample. In general, arrays are described as macroarrays or microarrays , the

difference being the size of the sample spots. Macroarrays contain sample spot sizes

of about 300 microns or larger and can be easily imaged by existing gel and blot

scanners. The sample spot sizes in microarrays are typically less than 200 microns in



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diameter and these arrays usually contains thousands of spots. Microarrays require

specialized robotics and imaging equipment that generally are not commercially

available as a complete system (Fig. 5)

In addition to Southern blots, gel electrophoresis, melting temperature

analysis with nonspecific dsDNA binding dyes, and probe based amplification

detection, microarrays have been used to analyze the specificity of PCR products.

DNA microarray technology (aka DNA chips or gene chips) involves the placement

of user defined oligonucleotides probes in specific locations on a solid substrate such

as glass. Following hybridization of target DNA sequences to probes anchored on a

chip’s surface, fluorescence detection can be used to monitor binding events.

Depending on the sensitivity required, microarrays can be used with or without

upstream amplification steps. Software analysis of large data sets that are generated

greatly facilitates the process of data analysis.

The advantages and limitations of several microarray software packages have

been reviewed. Microarrays may be an effective way of distinguishing between

nonspecific and target product formation and therefore this detection strategy may

allow the use of more primers in a multiplex PCR assay than would normally be

possible. Amplification methods have been used in combination with microarray

technology for the detection of E. coli O157:H7.77 Wilson et al.78 were able to

specifically detect 18 pathogenic microorganisms including, prokaryotes,

eukaryotes, and viruses using PCR in combination with a microarray containing over

50,000 probes and with a detection limit as low as 10 fg of DNA.



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Fig. 5 DNA microarray showing the steps in preparing oligonucleotide fragments that are subsequently

probed using complementary sequences for quantitative large-scale, high throughput screening of geneexpression using fluorescence.

Fluorescence in situ hybridization (FISH)

FISH is a technique for the probe-based identification of nucleic acids without

amplification. The technique can be used to specifically identify microbial cells in

environmental samples and rRNA molecules are frequently targeted. Fluorescently

labeled probes can be used to generate signals in the presence of specific targetsequences, seen with fluorescence microscopy. Typical steps include sample

preparation by fixation and permeabilisation, probe binding, removal of

unhybridized probes by washing, and flow cytometry or microscopy detection. A

FISH technique for the detection of Listeria monocytogenes showed specific detection

of the target microorganism and detection was possible in sheep milk samples.



22

Description of Probes and Their Development

The design and construction of gene probes requires careful scientific

experimentation and a series of complex decisions. A first step is to determine if the

gene probe is to be targeted to a particular pathogenic strain or to an entire

taxonomic group. A target must be chosen so that all of the microorganisms to be

detected contain such a gene. For probes designed to detect all members of a genus

or species, ribosomal RNA has been a popular target because it contains both

conserved and variable regions. If a pathogenic strain is sought, a probe is usually targeted to a virulence factor gene responsible for causing disease. A considerable

amount of research is needed to identify the genes involved and the role they play in

pathogenesis.



23

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Detection Final

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