1. Molecular diagnostics of infectious
diseases
The development and application of molecular diagnostic techniques has
initiated a revolution in the diagnosis and monitoring of infectious
diseases. Microbial phenotypic characteristics, such as protein,
bacteriophage, and chromatographic profiles, as well as biotyping and
susceptibility testing, are used in most routine laboratories for
identification and differentiation over the past several years. Advances in
molecular biology over the past 10 years have opened new avenues for
microbial identification and characterization. The traditional methods of
microbial identification rely solely on the phenotypic characteristics of
the organism. Bacterial fermentation, fungal conidiogenesis, parasitic
morphology, andviral cytopathic effects are a few phenotypic
characteristics commonly used. Some phenotypic characteristics are
sensitiveenough for strain characterization include isoenzyme profiles,
antibiotic susceptibility profiles, and chromatographic analysis of
cellular fatty acids.

2. Traditional Microbial Typing
Biotyping: Traditional microbial identification methods typically rely
on phenotypes, such as morphologic features, growth variables,and
biochemical utilization of organic substrates. The biologicalprofile of
an organism is termed a biogram. The determinationof relatedness of
different organisms on the basis of theirbiograms is termed biotyping.
For example, gram stain characteristics,indole positivity, and the
ability to grow on MacConkey mediumdo not aid in the
differentiation of nonenterohemorrhagic Escherichiacoli from E. coli
O157:H7. However, sorbitol fermentation hasproven to be an
extremely useful characteristic of the biochemicalprofile used to
differentiate these strains. Biograms of organisms are not entirely
stable, and severalisotypes may exist from a single isolate . It maybe
influenced by genetic regulation, technical manipulation,and the gain
or loss of plasmids. In many instances, biotypingis used in
conjunction with other methods to more accuratelyprofile
microorganisms.

3. • The antibiogramis the susceptibility profile of an organism to a
variety ofantimicrobial agents, whereas the resistogram is the
susceptibility profileto dyes and heavy metals. Bacteriocin typing is
the susceptibilityof the isolate to various bacteriocins, i.e., toxins
that areproduced by a collected set of producer strains. These three
techniques are limited by the number of agents tested per organism.
Antibiogram is the most commonly used to demonstrate relatedness,
Selective pressurefrom antimicrobial therapy may alter an
organism's antimicrobialsusceptibility profile. These alterations may
resultfrom chromosomal point mutations or from the gain or loss of
extrachromosomal DNA such as plasmids or transposons.
• protein analysis Commercially available antibodies are
routinely used to specificallyidentify antigenic proteins from a wide
variety of organisms .In some instances, the test may be used only to
identify thegenus and species of an organism.

4. • Electrophoretic techniques have been used to examineouter
membrane proteins, whole-cell lysates, and particularenzymes.
Outermembrane proteins and proteins from cell lysates are
examinedby SDS-PAGE electrophoresis.This technique denatures the
proteins and separates them onthe basis of molecular mass. The
protein profile may be usedto compare strain. The absence of a
particular protein may simply reflectdownregulation of that
particular gene product, rather thanthe loss of that particular gene.
Mutations that do not alter these characteristicswill not be detected
• CHROMATOGRAPHIC ANALYSIS: chromatographic analysis of short-
chain fatty acid productionis a routine method used to aid in the
identification of anaerobic bacteria. Computer-aided gas–liquid
chromatography is commerciallyavailable and is a means of
microbial identification. Many species have uniquecellular FA
chromatographic profiles. It is reliable when organisms are grown
under identical conditions and the cellular fatty acids areextracted
without technical variation.

5. • Nucleic acid amplification technology has opened new avenuesof
microbial detection and characterization suchthat growth is no
longer required for microbial identification.
Molecular methods have
surpassed traditional methods of detectionfor many fastidious
organisms. The polymerase chain reaction (PCR)and other recently
developed amplification techniques have simplifiedand accelerated
the in vitro process of nucleic acid amplification.The amplified
products, known as amplicons, may be characterizedby various
methods, including nucleic acid probe hybridization,analysis of
fragments after restriction endonuclease digestion,or direct
sequence analysis.
• DNA probes facilitate the identification of infectious agents that do
not grow rapidly.The diagnosis of infections in which the organisms
are not easily cultured or cannot be cultured at all . The technique
has been also used to monitor growth as an indicator of drug
resistance or to directly detect genes associated with antibiotic

6. Polymerase Chain Reaction
Nucleic acid analysis without amplification often has the
disadvantage of low sensitivity . PCR is the best-developed and most
widely used method of nucleic acid amplification. PCR is based on the
ability of DNA polymerase to copy a strand of DNA by
elongation of complementary strands initiated from a pair of closely
spaced chemically synthesized oligonucleotide primers.
Other Nucleic Acid Amplification Techniques:
Transcription-based amplification system (tas).
Described in 1989 by Kwoh et al., TAS includes synthesis of a DNA
molecule complementary to the target nucleic acid (usually RNA) and
in vitro transcription with the newly synthesized cDNA as a template
Nucleic acid sequence-based amplification ("NASBA"), or
transcription-mediated amplification (TMA) are other types of
techinques involving transcriptase enzyme.

7. • Ligase chain reaction (lcr): It is a probe
amplification technique first described in 1989 by Wu and Wallace.
Successful ligation relies on the contiguous positioning and correct
base-pairing of the 3' and 5' ends of oligonucleotide probes on a
target DNA molecule .
• Strand displacement amplification (sda): It is another non-PCR
nucleic acid amplification technique, developed in 1991. In this
system, DNA polymerase initiates DNA syntheses at a single-stranded
nick and displaces the nicked strand during DNA synthesis
• Analysis of Amplification Products:
Hybridization protection assay, dna enzyme immunoassay,
automated dna sequencing technology, rflp analysis .Viral load
determinations are currently used for evaluating HIV and
HCV disease by the use of PCR and bDNA technology .

8. • Molecular screening of particular at-risk populations for
group of possible pathogens is an exciting area of
development in molecular microbiology. The techniques being used
for molecular screening include the newer nucleic acid "chip"
technologies, multiplex PCR, and the use of broad-range PCR primers
and subsequent nucleic acid sequence analysis. "DNA chips.

9. FRAGILE X SYNDROME
• Fragile X syndrome is the most common inherited cause of mental
retardation. Fragile X syndrome affects approximately 1 in 4,000
males and 1 in 8,000 females. Women have two X chromosomes,
and a normal X chromosome may mitigate the effects of a defective
X chromosome. It is the most common cause of inherited mental
retardation .It is related to hyperexpansion and hypermethylation of
a polymorphic CGG trinucleotide repeat in the 5' untranslated region
of the FMR1 gene at Xq27.3..
• Symptoms
The effects of Fragile X on an individual's mental status are partially
related to the environment in which the individuals grows up, what
educational and therapeutic opportunities are available, and how
early those interventions are begun. The cognitive and
neuropsychological symptoms include

10. • Mild to severe mental retardation (IQ 20-80)
• Poor eye contact, hand biting, or hand flapping behaviors are present
in 16-25% of individuals. Some children with Fragile X syndrome may
also be diagnosed with autism, and some children with autism may
have undiagnosed Fragile X syndrome.
• Attention deficits, and in about 1/3 of males, aggression
• Approximately 20% of individuals have a seizure disorder
• Some musculoskeletal problems such as scoliosis may be present.
Adolescents and adults with Fragile X syndrome have a long thin face
with prominent ears, forehead, and jaw. A defect in the mitral valve
of the heart may also occur

11. • Diagnosis
Fragile X syndrome is difficult to diagnose in children. An affected
infant may develop normally at first. After age one year, the child
begins to have noticeable delays in language and short-term
memory. Physical signs of Fragile X, such as the typical facial
features, are only noticeable after the onset of puberty .
• Fragile X syndrome may be suspected if the individual has a number
of male relatives with mental retardation. However, in many families
the child with Fragile X syndrome is the first member known to have
mental retardation. If Fragile X syndrome is suspected, a blood test
known as FMR1/DNA is used to confirm the diagnosis.
• Southern blot analysis is the most commonly used method for
molecular diagnosis of FXS. A simplified strategy based on
fluorescent methylation-specific PCR (ms-PCR)and GeneScan

12. analysis for molecular diagnosis of fragile X syndrome. In this method
sodium bisulfite treatment is used to selectively modify genomic
DNA from fragile X and normal lymphoblastoid cell lines and from
patients. They performed ms-PCR amplification using fluorescently-
labeled primers complementary to modified methylated or
unmethylated DNA. Amplification products were resolved by
capillary electrophoresis. FMR1 mutational status was determined by
a combination of fluorescent peak sizes and patterns on the
GeneScan electropherogram . Recently, an immunocytochemical
test has been described to identify fragile X patients, based on
detection of FMR1 protein in cells using a monoclonal antibody
Prenatal diagnosis is possible Fragile X syndrome (FXS) is caused by a
change (mutation) in a gene on the X chromosome. . The gene on the
X chromosome that causes FXS is called the Fragile X Mental
Retardation 1 (FMR1) gene. The FMR1 gene makes a protein that is
needed for normal brain development. In FXS, the protein is not
made.