Lecture (1) Dr.
Walaa Alsharee
Diagnostic Microbiology
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MOLECULAR DIAGNOSTICS
Molecular diagnostics is growing rapidly. Molecular diagnostic tests detect
specific sequences in DNA or RNA that may or may not be associated with
disease, including single nucleotide polymorphism (SNP), deletions,
rearrangements, insertions and others. Clinical applications can be found in at
least six general areas: infectious diseases; oncology; pharmacogenomics;
genetic disease screening; human leukocyte antigen typing; and coagulation.
Each molecular assay requires three basic steps:
1. The extraction and purification of nucleic acid.
2. The amplification or making copies of the nucleic acid of interest (target) or
attaching multiple copies of a dye to a single target copy.
3. The detection of the amplified target using real time polymerase chain
reaction
(PCR) or end product detection including microarrays, Luminex (similar to
flow
cytometry), or sequencing.
Why Molecular Diagnostics Are Important
The medical community has recognized the importance of molecular
diagnostics for several decades, and this field is especially important to
cancer care. Molecular diagnostics have already improved cancer diagnosis
and treatment techniques, and research is continuing.
Why go for molecular way?
Traditional methods pose several
challenges
•Growth of fastidious pathogens
•Maintenance of viability
•Delay in cultivation
•Non-culturability of certain
organisms.
•Hazardous to propagate in lab.
•Cost versus clinical utility.
Disadvantages of Phenotyping
■Lack of Reproducibility
■Poor Discriminatory power
■Difficulties in Typing
■Genomic changes with Antibiotic resitance
patterns
■Technical manpower costs. (Developed World)
■
Advantage of Molecular Methods
▪ Aid in faster diagnosis of Diseases.
▪ Increased sensitivity and
specificity.
▪ Rapid detection of pathogen than
conventional methods.
▪ Decrease the man power need for
detection.
▪ Give rapid answers to treatment
options in life threatening
diseases.
▪ Adapted to instrumentation.
DNA VS RNA
❖ DNA AMPLIFICATION TESTS ARE KNOWN TO BE HIGHLY SENSITIVE AND SPECIFIC
FOR THE DIAGNOSIS OF OCULAR CHLAMYDIAL INFECTION, AND ARE SUPERIOR
TO OTHER LABORATORY METHODS SUCH AS TISSUE CULTURE, ANTIGEN
DETECTION, AND HYBRIDISATION. MORE RECENTLY, NEWER NUCLEIC ACID
AMPLIFICATION TESTS (NAATS) BASED ON AMPLIFICATION OF RIBOSOMAL RNA
(RRNA) HAVE BEEN DEVELOPED. AMPLIFICATION OF RRNA TARGETS PROVIDES A
POTENTIAL ADVANTAGE SINCE BACTERIAL RRNA IS PRESENT AT UP TO 10 000
TIMES THE COPY NUMBER OF GENOMIC DNA AND 1000 TIMES THAT OF
PLASMID DNA.
❖ THIS DIFFERENCE MAY BECOME ESPECIALLY IMPORTANT WHEN ORGANISMS ARE
PRESENT IN SMALL NUMBERS, SUCH AS IN ASYMPTOMATIC PATIENTS.
ALTERNATIVELY, BURTON, ET AL HAVE REPORTED THAT A HOMEBREW RRNA
AMPLIFICATION TEST IS FAR LESS SENSITIVE THAN DNA AMPLIFICATION.
MOLECULAR DIAGNOSIS
A. Identifying bacteria using 16S rRN
▪The 16S rRNA has conserved portions of the sequence.
▪Labeled probe specific for the 16S rRNA of a species
are added and then measured. This allows the
identification of Mycobacterium sp., Coccidioides
immitis, Histoplasma capsulatum.
▪Portions of the 16S rRNA are conserved across many
species and its amplification using primers allows
isolation and sequencing of the variable regions of the
molecules. These genus- or species-specific allows the
identification of pathogens that are impossible or
difficult to culture. Eg. Tropheryma whipplei the cause of
Whipple’s disease.
A
MOLECULAR DIAGNOSIS
B. TARGET AMPLIFICATION SYSTEMS
➢THE POLYMERASE CHAIN REACTION (PCR) IS USED TO AMPLIFY EXTREMELY
SMALL AMOUNTS OF SPECIFIC DNA.
➢THIS TECHNIQUE USES DNA POLYMERASES THROUGH ALTERNATE
CHANGES IN TEMPERATURE TO INITIATE REPLICATION IN EITHER THE 3’ OR 5’
DIRECTION. THE SPECIFICITY IS PROVIDED BY PRIMERS THAT RECOGNIZE A
PAIR OF UNIQUE SITES ON THE CHROMOSOME SO THAT THE DNA BETWEEN
THEM CAN BE REPLICATED.
➢PCR CAN ALSO BE PERFORMED ON RNA TARGETS, WHICH IS CALLED
REVERSE TRANSCRIPTASE PCR. THE REV TRANSCRIPTASE IS USE TO
TRANSCRIBE RNA INTO COMPLIMENTARY DNA FOR AMPLIFICATION.
➢PCR ASSAYS AVAILABLE COMMERCIALLY FOR CHLAMYDIA TRACHOMATIS,
NEISSERIA GONORRHOEAE, MYCOBACTERIUM TUBERCULOSIS,
CYTOMEGALOVIRUS AND ENTEROVIRUSES.
MOLECULAR DIAGNOSTICS
➢HYBRIDIZATION OF A CHARACTERIZED NUCLEIC ACID
PROBE (PRIMER, OLIGONUCLEOTIDES) TO A SPECIFIC
NUCLEIC ACID SEQUENCE IN A TEST SPECIMEN FOLLOWED
BY DETECTION OF THE PAIRED HYBRID.
➢THE NUCLEIC ACID PROBE TYPICALLY IS LABELED WITH
ENZYMES, ANTIGENIC SUBSTRATES, CHEMILUMINESCENT
MOLECULES OR RADIOISOTOPES TO FACILITATE DETECTION
OF THE HYBRIDIZATION PRODUCT.
Specimen Collection
! Preserve viability/nucleic acid integrity of
target microorganisms
! Avoid contamination
! Appropriate time and site of collection (blood,
urine, other)
! Use proper equipment (coagulant, wood, or
plastic swab shafts)
! Commercial collection kits are available
! The Clinical and Laboratory Standards Institute
(CLSI) has guidelines for proper specimen
handling
DNA extraction methods used in
research labs
Research
Lysis: grind in Liquid N2 and use detergent
Precipitation Part I: phenol/chloroform
extraction to get rid of proteins
Precipitation Part II: addition of salts to
interrupt hydrogen bonding between
water and phosphates on the DNA
Precipitation Part III: addition of ethanol to
pull DNA out of solution
Wash and resuspend: DNA is washed in
ethanol, dried, and resuspended in H20 or
TE buffer.
Overview of DNA Extraction
Break down Centrifuge to Precipitate
the cell wall separate the the DNA
and solids from using
membranes the dissolved isopropanol
DNA
Centrifuge to
separate the
DNA from
the dissolved
salts and
Dissolve Wash the sugars
DNA DNA pellet
with Ethanol
and dry the
pellet
16S rRNA Sequencing
CONTENT
1
Whatis16SrRNAgene?
2
Whatis16SrRNAsequencing?
3
Workflowof16SrRNAsequencing
16SrRNA
rRNA——molecular clock:
1.Universality
2.Activity in cellular functions
3.Extremely conserved structure and sequence
Three types of rRNA in prokaryotic
ribosomes:
•23S (3300 bp)
•16S (1550 bp) —— a standard in
bacterial taxonomic classification
•5S (120 bp)
16S rRNA:
⎫Risosomal RNA
⎫Phylogenetic markers
⎫1542 bp
The 16S rRNA gene consists of eight highly conserved regions and nine variable regions across
the bacterial domain. The degree of conservation varies widely between hypervariable regions,
with more conserved regions correlating to higher- level taxonomy and less conserved regions
to lower levels, such as genus and species.
Advantages
i.Universally distributed
ii.Abundance
iii.Capability to measuring phylogenetic
relationships across different taxa
iv.Horizontal gene transfer isn't a big
problem
v.Low costs
Table 1. Primer sets for the amplification of 16S rRNA. F=forward,R=reverse
Name of primer Sequence Name of Sequence
primer
8F AGAGTTTGATCCTGGCTCAG 785F GGATTAGATACCCTGGTA
27F AGAGTTTGATCMTGGCTCAG 805R GACTACHVGGGTATCTAATCC
336R ACTGCTGCSYCCCGTAGGAGTCT 806RB GGACTACNVGGGTWTCTAAT
337F GACTCCTACGGGAGGCWGCAG 907R CCGTCAATTCCTTTRAGTTT
337F GACTCCTACGGGAGGCWGCAG 928F TAAAACTYAAAKGAATTGACGGG
341F CCTACGGGNGGCWGCAG 1100F YAACGAGCGCAACCC
515FB GTGYCAGCMGCCGCGGTAA 1100R GGGTTGCGCTCGTTG
518R GTATTACCGCGGCTGCTGG 1492R CGGTTACCTTGTTACGACTT
533F GTGCCAGCMGCCGCGGTAA
*PROBE: A probe is a piece of DNA of the
sequence of the interest
*PROBE LABELLING: a labeled and defined
sequence use to search the mixture of nucleic acid
for molecule containing complementary sequence
*Probe concentration:
The higher the probe concentration, the faster
the hybridization occurs. However, the higher the
probe concentration, the higher the blot background
(i.e. probe will stick everywhere).
Low probe concentrations and long
incubations (overnight) usually produce the best
results.
*HYBRIDIZATION:
!*In molecular biology, hybridization is a phenomenon in
which single-stranded deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA) molecules anneal to complementary
DNA or RNA.
!*double-stranded DNA sequence is generally stable under
physiological conditions, changing these conditions in the
laboratory (generally by raising the surrounding
temperature) will cause the molecules to separate into
single strands.
!*These strands are complementary to each other but may
also be complementary to other sequences present in their
surroundings. Lowering the surrounding temperature allows
the single-stranded molecules to anneal or “hybridize” to
each other.
!*DNA replication and transcription of DNA into RNA both
rely upon nucleotide hybridization, as do molecular biology
techniques including Southern blots and Northern blots,the
polymerase chain reaction (PCR), and most approaches to
DNA sequencing.
*1.DNA-DNA HYBRIDIZATION:
THANK YOU :_)
