Recent advances in Microbiology

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Recent advances in Microbiology

• Dr.Mrs.N.ANBUMANI
• M.B.B.S,M.D.,PG Dip in BI., Ph.D
• ASSOCIATE PROFESSOR
• DEPARTMENT OF MICROBIOLOGY
• SRI RAMACHANDRA UNIVERSITY, PORUR,CHENNAI-600116.

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Introduction

• Clinical microbiologists have traditionally been
  concerned with the isolation and identification
  of pathogenic organisms from humans.
• Conventional methods involve isolating the
  organism of interest in pure culture and
  performing predetermined biochemical or
  immunologic tests to identify it.

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• In many respects, cultures and the adjunct
  methods used for identification are limited in
  sensitivity, specificity, or both.

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• To improve test sensitivity, shorten detection
  times, and identify hard-to-culture
  microorganisms, immunoassays were developed.
• These immunoassays allowed both large and small
  laboratories to expand services to meet
  diagnostic requirements in a timely fashion.
• The outlook for even more sensitive, more
  specific, and more rapid testing is currently
  being founded in the recent advances in molecular
  biology methods.

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• Historically, this analysis of pathogens has
  relied on a comparison of phenotypic
  characteristics such as biotypes, serotypes,
  bacteriophage or bacteriocin types, and
  antimicrobial susceptibility profiles.

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• This approach has begun to change over the past 2
  decades, with the development and implementation
  of new technologies based on DNA, or molecular
  analysis.

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• These DNA-based molecular methodologies include
  pulsed-field gel electrophoresis (PFGE) and other
  restriction-based methods, plasmid analysis, and
  PCR-based typing methods.

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• The incorporation of molecular methods for typing
  of pathogens has assisted in efforts to obtain a
  more fundamental assessment of strain
  interrelationship.

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CHARACTERISTICS OF TYPING METHODS

• There are a number important attributes for
  successful typing schemes the methodologies
  should be standardized, sensitive, specific,
  objective, and subject to critical appraisal.

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• All typing systems can be characterized in terms
  of typeability, reproducibility, discriminatory
  power, ease of performance and interpretation,
  and cost (in terms of time and money).

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Typeability

• Typeability refers to the ability of a technique
  to assign an unambiguous result (type) to each
  isolate.
• Nontypeable isolates are more common with
  phenotypic methods but can also occur with
  genotypic methods.

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Reproducibility

• The reproducibility of a method refers to the
  ability to yield the same result upon repeat
  testing of a bacterial strain.
• Poor reproducibility may reflect technical
  variation in the method or biologic variation
  occurring during in vivo or in vitro passage of
  the organisms to be examined.

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Discriminatory power

• The discriminatory power of a technique refers to
  its ability to differentiate among
  epidemiologically unrelated isolates, ideally
  assigning each to a different type.
• In general, phenotypic methods have lower
  discriminatory power than genotypic methods.

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Cost Ease of interpretation

• Most molecular methods require costly material
  and equipment but are relatively easy to learn
  and are applicable to variety of species.
• On the other hand, phenotypic methods also
  involve costs in labour and material and are
  restricted to a few species for example,
  antisera for Salmonella serotyping will not work
  to type gram-positive organisms.

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GENOTYPIC METHODS

• The goal of genotyping studies is that
  epidemiologically related isolates collected
  during an outbreak of nosocomial disease are able
  to be linked to one another.
• In other words, whether the isolates involved in
  a nosocomial outbreak are genetically related or
  not and thus originate from the same strain or
  otherwise

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• Therefore, the use of strain typing in infection
  control decisions is based on several
  assumptions
• (i) isolates associated with the outbreak are
  recent progeny of a single (common) precursor or
  clone,
• (ii) such isolates will have the same genotype,
  and
• (iii) epidemiologically unrelated isolates will
  have different genotypes

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• Typing of microorganisms classically involves
  the subdivision of a single or related species,
  using a set of defined characteristics

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Bacterial typing systems

• Typing methods fall into 2 broad categories
• Phenotypic
• Genotypic

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Phenotypic methods

• Biotype
• Antibiogram
• Serotyping
• Bacteriocin typing
• Phage typing

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Biotyping

• Based on subspecies diversity of
• Colony morphology
• Metabolic activity
• Toxin production

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Biotyping
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Antibiogram

• Changes in antibiograms may reflect spontaneous
  point mutations.
• Thus,isolates that are epidemiologically related
  otherwise genetically indistinguishable may
  manifest different antimicrobial susceptibilities
  due to acquisition of new genetic material over
  time or loss of plasmids

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Antibiogram
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Serotyping

• Uses a series of antibodies to detect different
  antigenic determinants on the surface of the
  bacterial cell
• The classic strain typing techniques
• It remains a key method for typing isolates of
  Salmonella,Shigella S.pneumococci

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Serotyping
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Bacteriocin typing

• Bacteriocin is protein products produced by other
  bacteria that inhibit growth of the test
  bacterium.
• Classifies bacteria according to their
  susceptibility to bacteriocin.
• Used in reference laboratories for typing
• K.pneumoniae P. aeruginosa.

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Bacteriocin typing
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Phage typing

• Classifies bacterial organisms according
• to susceptibility of the bacteria to lysis
• by the panel of bacteriophage.
• Phage typing has played useful in
• epidemiologic roles for S.aureus S.enterica
  serotype Typhi

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Phage typing
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Limitations of Phenotypic methods

• Influenced by environmental selective pressure
  -unstable antigenic traits
• -alterations in expression of
  
• traits being assessed
• Labour-intensive
• Impractical
• Slow
• Lack discriminatory power

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Genotypic methods

• Procedures based on DNA analysis offer a more
  stable and universal approach to typing
  microorganisms, and are used increasingly in
  microbiology laboratories to supplement
  traditional typing methods.

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Genotypic methods

• DNA typing differentiates organisms on the basis
  of genetic variation at the level of chromosome,
  plasmid or gene.

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Genotypic methods

• The total (genomic) DNA of a bacterium consists
  of a single chromosome (typically 0.6-10 megabase
  pairs Mbp, together with any plasmid DNA.
• In the case of fungi and protozoa, a number of
  chromosomes are present, in addition to
  mitochondrial DNA

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Genotypic methods

• Plasmid profile analysis PF
• REA of Plasmid DNA
• REA of Chromosomal DNA
• RFLP
• Pulse-Field Gel Electrophoresis (PFGE)
• PCR Amplification methods

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Plasmids

• Extrachromosomal genetic elements
• Number size of the plasmids are used as the
  basis of strain identification

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Plasmid profile analysisPF PLASMID FINGERPRINTING

• First molecular method to be used as a bacterial
  typing tool

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PF
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Pf

• Plasmid strain typing technique is successfully
  used for analysis of outbreaks of nosocomial
  infections community acquired infections
  especially by gram negative rods.

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Restriction Enzyme Analysis(REA)

• Restriction enzymes
• Makes double-stranded breaks in either Plasmid
• or Chromosomal DNA at specific nucleotide
• Locations Example
• EcoRI of Escherichia coli
• Recognizes GAATTC and cleaves between GA
• 2. HhaI of Haemophilus influenza
• GTPPyPuAC
• Pyany pyrimidine base Puany purine base
• Cleaves between PyPu

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REA

• Digest DNA using restriction enzyme(s)
• Separate the digested chromosomal
• DNA according to size in an agarose gel
• using gel electrophoresis
• Use molecular size marker
• Small DNA fragments migrate faster than large DNA
  fragments

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REA of Plasmid DNA

• Enhanced discrimination between organisms can be
  achieved through the cleavage of the plasmid DNA
  using restriction enzymes.
• These enzymes cut DNA at specific base sequences
  (restricted sites), and the frequency and
  location of the restricted sites determine the
  number of resulting fragments and their size.

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REA OF PLASMID DNA

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Limitations of plasmid REA

• 1.Plasmids can spread to multiple species of
  bacteria, causing a plasmid outbreak in which
  unusual antibiograms are recognised in multiple
  species.
• 2.The structure of individual plasmid the
  plasmid content of the strain may vary over time.

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REA of Chromosomal DNA

• Two methods of typing microorganisms by REA of
  chromosomal DNA
• Restriction enzyme that cuts the chromosome into
  hundreds of pieces (frequent cutter )followed by
  conventional electrophoresis
• Fragments of 25-50 kb are resolved

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REA of Chromosomal DNA (contd)

• 2. Restriction Enzyme that cuts the chromosome
  infrequently generating 10 to 30 bands followed
  by novel form of electrophoresis

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RFLP

• Chromosomal restriction digests produced by
  frequent cutting enzymes are separated by
  conventional agarose gel electrophoresis
• The DNA fragments are transferred onto
  nitrocellulose or nylon membrane
• The DNA on the membrane-is hybridized with a
  specific chemically or radioactively labeled
  piece of DNA or RNA( probe ) binds to few
  fragments complementary nucleic acid sequences.

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RFLP

• Variation in the number size of the fragments
  detected by hybridization are referred to RFLP
• VARIATION RIBOTYPING

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Advantage of RFLP

• Proportion of strains typable ALL
• Reproducibility excellent
• Discriminatory power moderate to excellent

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Limitations of RFLP

• Very difficult to interpret the complex profiles
  which consists of hundreds of bands that may be
  distinct or overlapping
• Ease of interpretation-moderate
• Ease of performance -difficult

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Pulsed Field Gel Electrophoresis

• PFGE first described in 1984 as tool for
  examining the chromosomal DNA
• Bacterial genome (2,500-5,000kbpairs in size)
• with rare restriction sites
• Restriction
  enzyme
• 10-30 restriction fragments (10-800kb)

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• Essentially all these fragments are resolved in
  to a pattern of distinct bands by PFGE
• By a specially designed chamber that positions
  the agarose gel between three sets of electrodes
  that form a hexagon around the gel

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PFGE

• PFGE is gold standard for bacterial sub typing
• Looks at whole genome of bacterial pathogens
  using rare cutting restriction enzymes
• 10-30 fragments ranging in size from 10kb to
• 800 kb in length are generated
• Larger pieces of DNA are separated by
• shifting direction of current frequently

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PFGE of E.fecalis
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PFGE of E.fecium
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PFGE of Ps. aeruginosa
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PFGE

• Proportion of strains typable ALL
• Reproducibility excellent
• Discriminatory power excellent

• Ease of interpretation-
• difficult
• Ease of performance -difficult

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Detection of a specific bacterial pathogen

• Cycling amplification technologies
• 1.1. PCR, real-time PCR and RT-PCR
• 1.2. Nested PCR
• 1.3. PCR-ELISA
• 1.4. Ligase chain reaction

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Polymerase Chain Reaction Kary Mullis invented
PCR
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DNA Between The Primers Doubles With Each Thermal
Cycle
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Molecular Beacon
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REAL TIME PCR
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REAL TIME PCR
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NESTED PCR
a modification of PCR intended to reduce the
contamination in products due to the
amplification of unexpected primer binding
sites.
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PCR-ELISA
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PCR-based Strain- typing methods

• Based on random sequences AP-PCR
• Based on heterogeneity restriction endonuclease
  sites AFLP
• Based on interspersed DNA repetitive elements
  REP-PCR

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Random Amplified Polymorphic DNA

• Williams et al. (1990) developed Random Amplified
  Polymorphic DNA (RAPD)
• Technique using very short 10 base primers to
  generate random fragments from template DNAs
• RAPD fragments can be separated and used as
  genetic markers or a kind of DNA fingerprint

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RAPD of E.faecalis
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RAPD

• Proportion of strains typable ALL
• Reproducibility GOOD
• Discriminatory power GOOD-HIGH

• Ease of interpretation-
• MODERATE
• Ease of performance - MODERATE

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AFLP

• Proportion of strains typable ALL
• Reproducibility GOOD
• Discriminatory power GOOD-HIGH

• Ease of interpretation-
• MODERATE
• Ease of performance - MODERATE

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rep-PCR

• This genomic fingerprinting method employed is
  based on the use of DNA primers corresponding to
  naturally occurring interspersed repetitive
  elements in bacteria, such as the REP, ERIC BOX
  elements, and the PCR reaction (rep-PCR).

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• Bacterial organisms carry certain genetic
  elements that jump, translocate, or transpose
  to new locations in the chromosome and called
  transposons or insertion sequences.
• If the copy number of IS elements is high enough,
  and if they are randomly distributed in the
  chromosome, DNA sequence between these elements
  can be amplified by PCR

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REP PCR of S.pneumoniae
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Limitations

• Patterns generated may be complex
• and difficult to interpret
• Technically demanding

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• Isothermal amplification technologies
• 2.1.Nucleic acid sequence-based amplification
• 2.2. Transcription-mediated amplification
• 2.3. Strand displacement amplification
• 2.4. Rolling circle amplification
• 2.5. Cycling probe technology
• 2.6. Branch DNA.
• 2.7. Hybrid capture.

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Nucleic acid sequence-based amplification (NASBA)

• an isothermal-based method of RNA amplification
  (Davey and Malek, 1989).
• RNA is amplified by the action of an enzyme
  cocktail that includes AMV Reverse Transcriptase,
  T7 RNA polymerase and RNAse H at a fixed
  temperature (41C).
• By coupling these technologies with a hand-held
  detection system, this method becomes a
  deployable monitoring device for onboard and
  remote sensing purposes.

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NASBA
By pairing this technique with the ability to
monitor the fluorescence signal produced from
Molecular Beacon probes (Figure) in real time as
they hybridize to the amplicon, real time
analysis of samples performed data in obtained
a matter of minutes
Figure 1. NASBA amplification pathway. Target
ssRNA (in this case, Noroviral genome) binds to
Primer 1. An RNA/DNA hybrid is formed by the
action of reverse transcriptase. RNaseH then
degrades the RNA component of the hybrid and
reverse transcriptase using Primer 2 makes a cDNA
of the target region. Because Primer 1 contains a
T7 RNA polymerase promoter, many copies of the
target RNA are made. NASBA reagents are available
from Biomerieux
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Transcriptionmediated amplification
TMA uses RNA transcription (RNA polymerase) and
DNA synthesis (reverse transcriptase) to produce
an RNA amplicon from a target nucleic acid.
Since RNA is more labile than DNA in the
laboratory environment, this feature diminishes
the possibility of carry-over contamination
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Rolling circle amplification
In rolling circle amplification (RCA), a single
forward primer is extended by DNA polymerase
along a circular template for many rounds,
displacing upstream sequences and producing a
long single-stranded DNA of multiple repeats.
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Cycling probe technology
Cycling probe technology (CPT) is an isothermal
probe amplification system for detection of
target DNA that utilizes a RNADNA chimeric probe
(RNA sequence flanked by two DNA sequences) to
hybridize to a specific region of an amplified
gene.
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Branched DNA
Another technique that utilizes signal rather
than target amplification is called branched DNA
(bDNA).
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HYBRID CAPTURE
Signal amplification is also the basis for some
commercial tests such as the Hybrid Capture
(HC2) assays from Digene (Gaithersburg, MD) The
chance of cross-contamination of reactions is
reduced.
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Detection of bacterial pathogens by multiple
targets or universal targets

• 3.1. Multiplex PCR
• 3.2. Microarray
• 3.3. Sequencing-based identification

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Multiplex PCR
Multiplex PCR utilizes more than one set of
primers in a reaction and can be used for the
simultaneous detection of multiple bacterial
pathogens.
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Multiplex PCR for Enterococcus
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Microarray

• Microarray refers to a small, two-dimensional
  high density matrix of DNA fragments which are
  printed or synthesized on a glass or silicon
  slide (chip) in a specific order.
• Hybridization of the DNA fragments to
  fluorescently labeled probes is detected by
  advanced instrumentation and software.

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MICROARRAY
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Pyrosequencing
Pyrosequencing ( AB, Uppsala, Sweden) is a
technology whereby a single-stranded DNA template
is prepared, a sequencing primer is hybridized to
a complimentary sequence on the template, and
enzymes catalyze a light reaction when each
nucleotide is incorporated into the growing DNA
strand .
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Sequencing-based identification

• Universal targets such as the 16S rRNA genes or
  the
• 16S23S rRNA gene interspacer region have been
  used extensively for bacterial identification,
  especially if bacteria are difficult to isolate
  by conventional methods
• Other universal targets such as heat-shock
  proteins, like hsp65 or cold-shock proteins can
  also be used

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Detection of bacterial pathogens by
non-amplification methods

• 4.1. Fluorescence in situ hybridization
• 4.2. Peptide nucleic acid-FISH
• 4.3. Line probe assay
• 4.4. Hybridization protection assay
• 4.5. Mass spectrometry

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Fluorescence in situ hybridization (FISH)

• Fluorescence in situ hybridization (FISH)
  assays use fluorescently labeled 16S rRNA or 23S
  rRNA probes and fluorescent microscopy to detect
  intact bacteria directly in clinical specimens,
  such as blood or tissue, or after enrichment
  culture.

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Line probe assay
The line probe assay (LiPA) consists of a
nitrocellulose strip with specific
oligonucleotide probes attached as discreet
parallel lines along the strip. Hybridization
results in a color change that can be detected
visually or by an automated reader.
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Hybridization protection assay

• Hybridization protection assays (HPA) utilize a
  chemiluminescent acridinium ester detector
  molecule on a DNA probe that targets the specific
  bacterial rRNA. The RNA/ DNA hybrid is detected
  in a luminometer.

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Mass spectrometry

• Mass spectrometry (MS) causes ionization and
  disintegration of a target molecule by bombarding
  it with electrons. The mass/charge ratio of the
  resulting molecular fragments is then analyzed to
  produce a molecular signature. MS has often been
  used to identify bacteria by protein signature

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Mass spectrometry
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Conclusion

• Molecular techniques do not substitute for
  conventional methods
• Each technique has its own unique advantages or
  disadvantages

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• The advantages of Nucliec acid detection tests
  over microbiologic methods include rapid results,
  low detection limits (theoretically a single
  cell), and specific organism detection.

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• contamination must be minimized,
• technicians must have proper training,
• and quality control procedures must be
  incorporated into routine laboratory workflow.

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• While the cost of some molecular diagnostic
  instruments is high,
• the benefits of faster turnaround time, high
  throughput, and enhanced sensitivity over
  traditional methods may override this obstacle.
• Another important consideration is that
  laboratory space must be allocated for
  instruments and dedicated as DNA-free areas.

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