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molecular_methods_in_diagnosis_of_infectious_diseases

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									Molecular Diagnosis of Infectious Diseases

Dr. Kavitha Santhosh, MD.

Broad Classification
Molecular Methods

Lipids

Proteins

Nucleic Acids

Why go the Molecular way?
Traditional methods pose several challenges  Growth of fastidious pathogens  Maintenance of viability  Delay in cultivation  Poor reliability  Non-culturability of certain organisms

What’s the advantage of molecular methods
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Aid in diagnosis of infectious diseases Increased sensitivity and specificity Rapid detection than conventional methods Give rapid answers to treatment options in life threatening infections

Molecular methods are necessary if the traditional methods provide poor results
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Microscopy gives false positive results - T.vaginalis,

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N.gonorrhoeae Intracellular pathogens – viruses Low sensitivity– Chlamydia sp.,Neisseria sp. Seropositivity is common – Chlamydia sp. Subtyping is mandatory – HSV, HPV, HCV Microbial growth is slow – M. tuberculosis

Molecular Methods

Signal amplification
• Nucleic acid probes

Post amplification analysis
• Gel electrophoresis • Sequencing

• Hybrid capture
• In situ hybridization

Nucleic acid amplification
• Polymerase chain reaction • Ligase Chain Reaction • NASBA • SDA

Molecular Methods

Signal amplification •Nucleic acid probes • Hybrid capture • In situ hybridization

NUCLEIC ACID PROBE & HYBRIDIZATION

NUCLEIC ACID PROBE & HYBRIDIZATION

Hybridization : Ability of 2 nucleic acid strands that have complementary base sequences to specifically bond with each other & form a double stranded molecule (duplex or hybrid) Probe: Nucleic acid strand from an organism of known identity.Usually a single stranded DNA or RNA Conjugated probe : ( radio active, enzyme etc) Target: Nucleic acid strand of the organism to be detected or identified Duplex: (positive hybridization) Duplex can be DNA-DNA, DNA-RNA & even RNARNA depending upon the design of the assay

HYBRIDIZATION STEPS & COMPONENTS 1. Production & labelling of single- stranded probe nucleic acid 2. Preparation of single stranded target nucleic acid 3. Mixture & hybridization of target & probe nucleic acid 4. Detection of hybridization

I. Production & labelling of singlestranded probe nucleic acid : Probe design Depends on the sequence of intended target nucleic acid (ie intended use) Probes can be chemically synthesized using instrumentation, commercially The user need to supply the manufacturer with the desired nucleotide base sequence

II. Preparation of target nucleic acid  Target nucleic acid must be single stranded & its base sequence integrity should be intact Target preparation steps 1. Enzymatic/chemical destruction of the microbial envelope to release the target nucleic acid 2. Stabilization of target nucleic acid to preserve structural integrity 3. If target is DNA, denaturation into single strand ( generally heated to 94 C)

III. MIXTURE & HYBRIDIZATION OF TARGET & PROBE Environment in which probe & target are brought together is important Hybridization stringency is most affected by
A. Salt concentrations in hybridization buffer B. Temperature C. Concentration of destabilizing agents

IV. Detection of hybridization Depends on the reporter molecule used for labelling probe nucleic acid 3 main reporter system used are 1. Radioactive reporter 2. Biotin-avidin reporter 3. Chemiluminiscent reporter

Nucleic acid hybridization: Summary Release of nucleic acid from specimen Denaturation (dsDNA)

Hybridization with probe
Detection of hybrid

Nucleic acid probes
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One of the earliest and most common methods. Easy and direct detection from specimens. Use of labelled probes Sensitivity and specificity : 90 - 100%
Organisms being detected
1. 2. 3. 4. 5. 6.

N. gonorrhoea C. trachomatis M. tuberculosis C . jejuni L. monocytogenes B. burgdorferi

Hybridization techniques:

Solution format Solid support format
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Filter hybridizations Southern hybridizations Sandwich hybridizations In situ hybridizations

SOLUTION HYBRIDIZATION FORMAT

SOLID SUPPORT HYBRIDIZATION FORMAT

1.Southern blot hybridization 2.Filter hybridization 3. Sandwich hybridizations

SOUTHERN HYBRIDIZATION

Southern blot/ DNA blot



Southern blot from a clinical sample

Uses of Southern blotting:
  

Identify the organisms Detect mutations To type the strains for epidemiological investigations

In situ hybridization  Allows a pathogen to be identified within the context of the pathologic lesion being produced  Uses patient cells or tissues as the solid support phase  Combines the power of molecular diagnosis with the additional information that histopathologic examination can provide

In situ hybridization
Molecular pathology
1.

2.

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FISH (fluorescent labeled probe) CISH (chemiluminiscent probe) Target: r RNA (indicative of viability)
Organisms detected:

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H. pylori, T. whipplei, L. pneumophila M. avium (16s RNA), M. leprae

Molecular Methods

Nucleic acid amplification

• PCR
• LCR • NASBA • SDA

WHY AMPLIFICATION??
If sufficient target nucleic acid is not present in the reaction, hybridization can give false negative results. To circumvent this, nucleic acid amplification is used How to amplify the nucleic acid? By allowing repeated replication of target nucleic acid

Nucleic acid amplification
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Widely used Amplifies target nucleic acid Useful when the load of the organisms is low Speedy, sensitive

POLYMERASE CHAIN REACTION
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Combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication that are applied repeatedly through numerous cycles. By this method, a single copy of nucleic acid target, often undetectable by standard hybridization methods, is multiplied to many copies within a relatively short period This provide ample target that can be readily detected by numerous methods

Polymerase chain reaction
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Basic steps: Denaturation of the target (dsDNA)
Annealing of primers Extension of primer-target Duplex Detection of PCR products

Extraction & denaturation of target nucleic acid
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
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Nucleic acid extracted from the organism or clinical sample by heat, chemical or enzymatic methods. Denaturation of dsDNA to a single strand is accomplished by heating to 94 C Denaturation not necessary for RNA Once extracted, target nucleic acid is added to the reaction mix that contains all necessary components of PCR to occur (primers, covalent ions, buffers, enzymes etc)

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PRIMER ANNEALING Primers are short single sequences of nucleotides (oligonucleotides) Selected specifically to hybridize (anneal) to a particular nucleic acid target, essentially functioning like probes Primers are designed to be used in pairs that flank the target sequence of interest

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PRIMER ANNEALING contd… When the primer pair is mixed with the denatured target DNA, one primer anneals to a specific site at one end of the target strand, while the other primer anneals to a specific site at the opposite end of the other, complementary target strand Once the duplexes are formed, the last step in cycle which mimics the DNA replication process, begins.

DENATURATION & PRIMER ANNEALING

Extension of primer-target duplex Annealing of primers to target sequence provides the necessary template format that allows DNA polymerase to add nucleotides to 3’ end of each primer & produce by extension a sequence complementary to target sequence

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

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TAQ POLYMERASE is the enzyme commonly used for primer extension, which occurs at 72 C It can function efficiently at elevated temperature & withstand the denaturing temperature of 94 C through several cycles All the 3 reaction components of PCR occur in the same tube that contains a mixture of target nucleic acid, primers, deoxynucleotides, buffer, Mgcl2, salt)

THERMAL CYCLER  To maintain continuous reaction cycles, programmable thermal cyclers are used



For each target sequence originally present in the PCR mixture, 2 double stranded fragments containing the target sequence are produced after one cycle

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At the beginning of the second cycle of PCR, denaturation then produces 4 templates to which the primers will anneal. Following extension at the end of the second cycle, there will be 4 double stranded fragments containing target nucleic acid After 30 to 40 cycles, 107 to 108 target copies will be present in the reaction mixture

Detection of PCR products (Post amplification analysis)
Gel electrophoresis is the most common method Any of the basic methods previously described for detecting hybridization can also be adapted

GEL ELECTROPHORESIS


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A portion of PCR mixture after amplification is subjected to gel electrophoresis After electrophoresis, the gel is stained with ethidium bromide to visualize the amplicon Using molecular weight size markers, the presence of amplicons of appropriate size is confirmed.

GEL ELECTROPHORESIS

Polymerase chain reaction


Rare organisms detected:
 

Rickettsia C. trachomatis

Polymerase Chain Reaction

Types of PCR ■ Nested PCR  Broad range PCR  Multiplex PCR  RT - PCR  Real Time PCR

Nested PCR
 

Two primers added sequentially First amplicon serves as a target for second amplification
Disadvantage: amplicon contamination Organisms detected: M. tuberculosis C. trachomatis L. pneumophila H. pylori

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Broad-range PCR
Organisms being detected
M. Tuberculosis B. henselae T. whipplei
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Use of broad range specificity primers Advantage: Primers target a larger group of microorganisms Disadvantage: detection of phylogenetically related organisms but not those in the group of interest

Multiplex PCR
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Multiple primers Each primer can target a different organism Used to detect: • Viral agents causing encephalitis (HSV, Enteroviruses, West nile viruses)  Bacterial agents causing meningitis (N. meningitidis ,M. tuberculosis etc)  Enteric pathogens (Salmonella campylobacter ,Shigella,E.coli

Reverse – transcriptase PCR
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Target - RNA Reverse transcriptase makes a DNA copy of target DNA polymerase makes a dsDNA Routine PCR technique is done Useful for detecting RNA viruses (eg:- HIV, Hepatitis B virus etc)

Real Time PCR
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Quantification & detection simultaneously with amplification Detection of amplicon in real time Methods to quantify amplicons
Nonspecific methods - SYBR green  Specific methods 



Organisms detected: L. pneumophila

Organism Assay

C. HCA trachomatis
PCR Culture

Sensitivit y 93.3%
97.8% 73.3% 100%

Specificity
100% 99.3% 100% 99.4%

N. HCA gonorrhoea
PCR
Culture

100%
90.5%

99.0%
100%

OTHER MOLECULAR METHODS Ligase chain reaction Nucleic acid sequence based analysis Standeard displacement analysis DNA microarrays

Molecular Methods helps in DIAGNOSIS, predicting PROGNOSIS and tailoring THERAPY to your patients needs.
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They have there limitations and you need to understand their probabilistic nature
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Expense Expertise

“Remember that taking a Good History allows you to get a diagnosis in 80-90% of cases without the Need of using Fancy technology”

“It was the first day of the rest of my life”!! Karry Mullis,about the day he had the first solid idea about developing PCR


								
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