International Journal of Livestock Research ISSN 2277-1964 ONLINE www.ijlr.

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Vol 2(1) Jan12

Use of DNA Technologies for the Diagnosis of Bacterial Diseases

Radhika Syam*, Justin Davis K, Pratheesh M D, Remya V and Anu Gopal
Indian Veterinary Research Institute, Izatnagar, Bareilly- UP
*Corresponding author - PhD Scholar, Veterinary Bacteriology and Mycology Division Indian
Veterinary Reasearch Institute (IVRI) Bareilly, UP-243122

Abstract
Timely diagnosis is a major hurdle in infectious diseases. Diagnosis cannot be awaited for satisfying
Koch postulates; there comes the importance of DNA technologies. These methods can also be used for
diagnosis of unculturable and non viable organisms rapidly. These methods are easy to be done and
automation reduces labor.Applications of DNA technologies for diagnosis of several bacterial diseases
are explained in this article.

Keywords: DNA technologies, bacterial diseases

Introduction
Bacteria have existed long before mammalian evolution and infectious diseases have been
present as long as there have been humans. Historically, treatment of infectious diseases was
poor due to inadequate knowledge, a limitation that substantially contributed to disease spread
and massive death. Approximately 150 years ago Louis Pasteur formulated the germ theory,
explaining how contagious diseases could spread between humans and Robert Koch gave the
first proof of the germ theory by demonstrating how Bacillus anthrax could infect sheep and later
discovered the causative bacteria of tuberculosis and cholera. Until the discovery of the duplex
DNA structure and the complementary rules in 1953 that the era of nucleic acid-based
molecular diagnostics of infectious diseases began.
The cornerstone of most molecular biology technologies is the gene. To facilitate the study of
genes, they can be isolated and amplified. Nucleic acid-based diagnostics of infectious diseases
involves detection and characterization of both bacterial and viral infection using DNA/RNA
methods. Today the major driving forces for developing new diagnostic techniques are reduced

some bacterial pathogens like Neisseria gonorrhea, are difficult to be cultured in lab condition.

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There are two aspects of disease diagnosis with DNA technology. Those designed to identify

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hands-on-time and faster methods, as well as increased sensitivity.Classical cultivation based

identification and monitoring methods have dominated various clinical microbiology labs, but

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specific pathogen responsible for causing disease and those test which are designed to identify
genes coding for virulence factors of a bacteria. (Caskey, 1987)
Detection of Pathogens
1. Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) was developed approximately 25 years ago (Saiki et al., 1985
and1988). In PCR, DNA is amplified in a thermocycler by repeating the three major steps:
denaturation of DNA template into single stranded DNA, annealing of oligonucleotide primers to
their complementary targets, and extension of the primers by DNA polymerase to generate a
copy of the target gene (Yang and Rothman, 2004). PCR has allowed a spectrum of advances
ranging from the identification of novel genes and pathogens to the quantification of
characterized nucleotide sequences (Erlich et al., 1991). PCR assays rapidly and precisely
detects the presence of microorganisms, including those that are fastidious and slow growing,
directly from clinical specimens. PCR is also used to test for the presence of antimicrobial
resistance genes in samples. Numerous different PCR-based assays have been developed for
diagnosis of infectious diseases.
A.

Specific PCR

Specific PCR is the simplest PCR approach which is designed to amplify a gene specific
to a target microbe. So the primers in specific PCR are designed such that they are strictly
specific for the target microorganism. This is the most widely used method in the diagnosis of
infectious disease. Many organism such as Mycobacterium, Streptococcus, Pneumococcus,
Borrelia, Helicobacter, Rickettsia, Ehrlichia, Coxiella can be directly detected in clinical
samples.Tuberculosis caused by Mycobacterium tuberculosis requires a fast, sensitive and cheap
detection technique in order to initiate adequate control measures in order to contain the disease
(Palomino, 2005). Multiplex PCR followed by hybridization to a DNA strip for detection of
multidrug resistant M. tuberculosis is commercially available, for the simultaneous detection of

phase of the disease (de Abreu et al., 2006).

B.

Real time PCR

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PCR and serological tests has improved the sensitivity of the diagnosis of leptospirosis in early

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rifampin (rpoB) and isoniazid (katG) resistance (Hillemann et al., 2005). The combined use of

International Journal of Livestock Research ISSN 2277-1964 ONLINE www.ijlr.org

Real Time PCR is simple, specific, sensitive and fast.

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Real time PCR comprises both

amplification and fluorescent detection of the sample in the same step. This also reduces the risk
of contamination with amplified nucleic acids because the analysis is performed in a closed
vessel. In general there are two different detection methods for real time PCR. One method relies
on a fluorescent stain (e.g. SYBR Green), which is specific for double stranded DNA, and the
other method relies on fluorescent resonance energy transfer (FRET) probes. SYBR Green
provides a sensitive, but not specific, signal, whereas the FRET probes provides a sensitive and
specific signal. Three different FRET probes exist: 5 nuclease probes (TaqMan), molecular
beacons, and FRET hybridization probes. TaqMan probes carry both a fluorescent dye and a
quenching dye, and after the amplification step the 5 nuclease activity of the Taq polymerase
separates the fluorescent dye from the quencher, resulting in an increasing abundance of
fluorescence after each PCR cycle (Espy et al., 2006).
Molecular beacons is a probe embedded within two complementary sequences, and carry both a
fluorophore and a quencher at the ends of the probe, and in the absence of a target the probe
exist as a hairpin structure, forcing the quencher near the fluorophore. When the probe hybridizes
with a complementary target sequence, the fluorescent dye and the quencher is separated, so that
a fluorescent signal is generated (Tyagi and Kramer, 1996). FRET hybridization probes consist
of two DNA probes, each carrying a fluorophore. The upstream probe carries the fluorescent dye
at the 3 end, and a second probe designed to hybridize downstream, carries an acceptor dye at its
5 end and is phosphorylated at its 3 end to prevent it from being used by Taq polymerase during
PCR amplification. The fluorescence from the upstream probe is absorbed by the adjacent
acceptor dye at the downstream probe when the probes are hybridized next to each other and the
dye is excited and emits light with a third wave length which is detected .
The limitations of real time PCR are the possibility of inhibition of the polymerase by the
presence of certain compounds, and the risk of detecting contaminating DNA due to the high
sensitivity of the method (Valasek and Repa, 2005). Staphylococcal methicillin resistance is

C.

Broad Range Ribosomal PCR

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inactivated by -lactams) is detected by the above method(Livermoore, 2006).

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mainly mediated by the mecA gene, which encodes a peptidoglycan transpeptidase (not

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Broad range ribosomal PCR has played a key role in the identification of novel bacterial
pathogens. The prokaryotic small subunit has RNA with a sedimentation coefficient of 16S has
been most used for bacterial characterization as it contains alternating regions of sequence
conservation and heterogenity. This procedure employed primers that recognize the conserved
regions of 16S rRNA genes, amplification and sequence comparison with the known
sequence in databases to characterize the bacteria. These characteristics inspired Woese in 1987
to deem rRNA the ultimate molecular chronometer (Woese, 1987). The first agent discovered
by this technique was the agent of Bacillary angiomatosis in AIDS patients by Rochalimaea
quintana. Often partial sequencing of the 16S rRNA gene is enough to identify bacteria, and it
has been shown that sequencing of the 5 end of 16S rRNA is sufficient for species level
identification of most clinically relevant Mycobacterium isolates (Rogall et al 1990; Patel, 2001).
D.

Rapid Primer PCR (RAPID) PCR

In RAPID PCR random range of nucleotide hexamers are used as primers. Being
hexamers, these primers binds to multiple sites on bacterial chromosome, producing multiple
sizes of amplicons. After running the PCR products on gel by electrophoresis a pattern of
bands are obtained. RAPID-PCR can easily distinguish between different range of a bacterial
species. Rapid PCR technology is used for identification of infectious organisms, determining
antibiotic resistance eg, vanA and vanB genes for vancomycin-resistant Enterococci (VRE),
detecting genes and gene mutations.Sensitivity equals and frequently exceed culture, enabling
detection of <10 copies of target nucleic acid per reaction. It also eliminates contamination
problems seen with PCR.
E.

Nested PCR

In Nested PCR genomic DNA is amplified with two sets of primer. Firstly the target sequence is
amplified using first set of primers, and a portion of amplification product is reamplified
using another pair of PCR primers complimentary to the regions located beyond the 3' end
of first primers. As a result the second PCR product is shorter than first set. This method can be

infection in patients with severe pneumonia. (Talkington et al., 1998). Single-tube nested PCR in

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nested PCR specific for the Mycoplasma pneumoniae P1 gene was used to diagnose mycoplasma

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used to increase the sensitivity of detection and reduce contamination of nonspecific products. A

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the diagnosis of tuberculosis for the repetitive IS6110 sequences was formulated by Chan et
al.(Chan et al., 1996)
2. Ligase Chain Reaction
Ligase chain reaction (LCR) is a different type of amplification reaction in which the
clinical sample is added to a reaction mixture containing a thermostable DNA ligase, a vast
excess of two oligonucleotide primers specific to the pathogen to be detected and NAD
(Nicotinamide adenine dinucleotide). If the sequence alignment is perfect, the ends of the primer
will be close enough the each other to be covalently sealed by the DNA ligase. As in PCR, the
denaturation step leads to separation of primers from the test DNA and then unused primer can
bind to DNA and be linked. With the repeated cycles of denaturation, annealing and DNA ligase
action, primer pair increases in concentration if they bind tightly enough to target DNA sequence
for joining by DNA ligase. Finally, a wash step removes unhybridized primers and then a final
denaturation step removes the bound primers and primer pairs from the test DNA. There will be
two band on gel electrophoresis; one band corresponding to probe used for amplification,
a second band equal to size to the sum of sizes of two probes, LCR is highly efficient as it can
detect as few as 200-300 target molecule in a samples. LCR procedure allows an automated
detection by employing flourescence or hapten labelled probe.LCR is used for detection of
Borrelia burdgoferri (Hu et al.,1991), Listeria monocytogenes (Wiedmann et al., 1992),
Neisseria gonorrhoeae (Birkenmeyer and Armstrong,1992), Mycobacterium tuberculosis
(Iovannisci and Winn-Deen,1993) and Chalymdia trachomatis (Dille et al.,1993)
3. Nucleic Acid Hybridization
In Nucleic acid hybridization technique, small 15-30 bp long nucleotide sequence are used
to detect complementary sequence in nucleic acid samples. Probes are prepared synthetically and
can be radioactivity or non-radioactively labelled. Probes are being utilized in clinical diagnosis
for detection of microorganism in various samples. First the DNA is isolated from the test
sample and subjected to southern blot or dot blot hybridization with the probe. Some bacterial

4. Fluorescent In situ Hybridization

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spp. Campylobacter spp. etc.

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agents against which probe are used for diagnosis are Legionella, M.tuberculosis, Chlamydia

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Fluorescent in situ hybridization (FISH) is a tool that today is widely used for identification,
visualization and localization of microorganisms in many fields of microbiology (Peters et al.,
2006; Poppert et al., 2002; Thurnheer et al., 2005). In 1989, De Long et al. reported the first in
situ hybridization using fluorescently labeled ribosomal RNA-directed oligonucleotide probes. It
allowed simultaneous monitoring of different species in the same sample through the use of
multiple probes labeled with different fluorescent dyes. it is a safer method, gave better
resolution and higher sensitivity. FISH is a rapid method for visualization and identification of
bacteria directly in samples of interest and the technique is able to reveal non culturable species
(Amann et al., 2001). Sogaard et al. used peptide nucleic acid (PNA) probes for FISH on
blood samples in order to rapidly detect a series of infectious bacteria (Sogaard et al., 2005).
In a study by Poppert et al. real time PCR and FISH were evaluated as rapid diagnostic
techniques for determination of Neisseria meningitides directly in cerebrospinal fluid (Poppert et
al., 2002).
5. DNA Microarrays
Bacterial DNA microarrays for clinical microbiology build upon the increasing amount of
sequence information available on pathogenic bacteria. Basically DNA microarrays are dot blot
hybridization experiments performed in a small and highly parallel format, allowing
hybridization to multiple targets in the same assay. The amount of hybridized sample is
quantified by signal intensity (Cassone et al., 2007). Korczak et al. in 2005 had surveyed
pathotypes of different Escherichia coli strains and found a total of 32 probes that could
distinguish the different pathotypes. Kang et al. in 2006 used

DNA

microarrays

suppression subtractive hybridization to determine the complexity of Salmonella

and

enterica

species and define genetic traits that are characteristics for epidemic strains of S. enterica.
6. Ribotyping Or Insertion Sequence Typing Using RFLP
In this method, DNA from the bacterial strain to be identified is isolated and is digested
with one or more restriction enzyme. The fragments are separated on a gel by

of restriction enzyme chosen, may be species or strain specific. Only disadvantage of this
method is large amount of nucleic acid sample is required.

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probe that hybridizes to rRNA genes. So the pattern of bands depending upon the type

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electrophoresis and is transferred to a membrane filters. The filter is incubated with a

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Identifying Virulence Factors

1)

Cloning

In this approach gene from the virulent strain of pathogen of interest is cloned into strain of E.
coli that is avirulent and look for genes that increases the virulence of E. coli. Another way of
identifying virulence factors is cloning and expression of genes responsible for virulence in
E. coli and studying the immunological and immunodiagnostic potential of the expressed protein.
2)

Transposon Mutagenesis

Transposons can be used for insertional mutagenesis to generate a collection of mutants. These
transposon also carry along with them a selectable marker, such as antibiotic resistance
gene. A transposon integrating in a gene of Salmonella strain responsible for adhesion, so
bacterial will no more be able to attach to tissue culture cells. Integrated transposon due to its
selectable marker makes the identification of gene very easy. Only problem is that the
transposon frequently carry transcriptional terminators and leads to polar mutations i.e.,
inhibition of expression of downstream genes also.
3)

Using Transcriptional Fusion Approach

In transcriptional fusion approach, promoter/operator (regulatory region of virulent gene) of a

virulent gene is fused to reporter gene like lacZ, uidA, cat etc. Thus the reporter gene is
expressed in the condition virulent gene would normally been expressed. It makes the
identification of gene and also the conditions of cellular machinery and environmental factors
(e.g. temperature dependence) for its expression.
4)

In vivo Expression Technology (IVET)

It is now possible to identify genes that are expressed by bacteria only in host species
using in vivo expression technology, where virulence genes that are transcriptionally induced
during infection can be identified (Mahon et al., 1995).It is observed that purine auxotrophs of
serovar Salmonella typhimurium is unable to infect mice. Promoter less purine biosynthetic gene

which are surviving have expressed a gene particularly in vivo.

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inoculated into an animal and surviving clones are isolated. It is presumed that clones of bacteria

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is introduced into chromosome of a strain randomly, the collection of fusions is

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5)

Vol 2(1) Jan12

Signature Tagged Mutagenesis

It is also an in vivo form of transposon mutagenesis involving cloning of random oligomers into
a transposon. Thus a transposon has a signature tag on to it. These mixture of transposon are
transformed into the target bacterial strain and transformations are selected by antibiotic
resistance. Now these bacterial isolates are injected into animal for infection development. After
development of infection, PCR based amplification is done based on primers which
recognize the transposon portion as it is present in all the transformants, and tags are
amplified. Now these mixture of tags are used to probe original collection of transposon
generated mutants. So any loss of a mutant during the infection process can be known with this
technology.
Future Perspectives
Detection of infectious bacteria by use of molecular diagnostics is an emerging
technology but still it is not mature enough to have ripened fruits i.e., entire replacement of
traditional culture methods. These methods will be automated so that they require little handson-time, are easy to use, and will allow detection of several different pathogens in the same
analysis. Recombinant DNA technology has rapidly expanded our ability to diagnose
disease.The wide application of recombinant DNA diagnostics will depend on simplicity, speed
of results, and cost containment.

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Caskey CT. 1987. Disease Diagnosis by Recombinant DNA Methods. Science.236 4806: 12231229
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Chan CM, Yuen KY, Chan KS, Yam WC, Yim KH. Ng WF and Ng MH. 1996. Single-tube
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de Abreu FC, Teixeira d FV and Calo RE. 2006. Polymerase Chain Reaction In Comparison
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