Molecular Diagnostics (Phân tích nhanh chất lượng thực phẩm)

Diagnosis of infectious diseases Culture methods  Immunological methods antigen detection, antibody detection: specific interactions between antibody and antigen  Nucleic acid-based m

Some success stories

Glucose sensor

→ Diabetic patients can monitor blood glucose at home

Glucose sensor

Pregnancy testing

 Human chorionic gonadotropin (hCG) is a glycopeptide

hormone produced by the placenta during pregnancy

 The appearance and rapid rise in the concentration of hCG in the woman's urine makes it a good pregnancy marker

 Usually, concentration of hCG in urine is at least 25

mIU/ml as early as seven to ten days after conception

 The concentration increases steadily and reaches its maximum between the eighth and eleventh weeks of pregnancy

Pregnancy testing

Molecular diagnostics

 Genetic identification

Characteristics of a detection system

 A good detection system should at least have 3 qualities:

• Sensitivity

• Specificity

• Simplicity

very small amounts of target even in the presence of

other molecules

target molecule only.

inexpensively on a routine basis.

Diagnosis of infectious diseases

 Viruses (HIV, HBV, HCV…)

 Bacteria (Vibrio cholerae, Mycobacterium tuberculosis…)

 Fungi (Aspergillus, Candida)

Diagnosis of infectious diseases

 Culture methods

 Immunological methods (antigen detection, antibody

detection): specific interactions between antibody and

antigen

 Nucleic acid-based methods (PCR, NASBA, LAMP):

specific binding between DNA or RNA molecules

Culture methods

 Use media for isolation and identification of pathogenic organisms Useful for antimicrobial sensitivity test

 Different types of media:

• Enriched media: high nutritive value to promote growth

• Selective media: allow only needed bacteria to growth

• Indicator media: to distinguish one microorganism type from another growing on the same media

Culture methods

 Evaluation based on:

• Colony morphology (shape, size, color, edge of colony)

• Pigment production

• Hemolytic activity

• Plaque formation, cytopathic effect for virus

Culture methods

 Advantages: gold standard method

 Disadvantages: laborious, time-consuming, need of special facilities, risk of infection…

Culture methods

Blood-free, charcoal-based selective medium agar for isolation of

Campylobacter

Culture methods

Blood agar plates are often used to diagnose infection On the

right is a positive Streptococcus culture; on the left a positive

Staphylococcus culture.

Culture methods

Viral Plaques of Herpes Simplex Virus

IMMUNOLOGICAL METHODS

 Microbial antigen detection → direct evidence of infection

 Antibody detection → indirect evidence of infection

 However, not all infectious agents have available antigen assay → detection specific antibodies is useful

Antigen or antibody detection?

Immune system: a quick overview

Analysis of HBV infection

Analysis of HBV infection

ELISA (Enzyme Linked Immunosorbent Assay)

ELISA to detect antigen

 capture antibody, directed against

antigenic protein, is linked to a solid support

 clinical sample is added, the antigen, if

present, will be capture by the bound antibody

 washing to remove unbound molecules

 addition of second antibody which will be bind to the antigen This

antibody is linked to an enzyme

 washing to remove unbound molecules

 addition of a colorless substrate → color product → positive result

ELISA to detect antibody

 viral protein is linked to a solid support

 clinical specimen is added The antibody

against the virus, if present, will bind to the immobilized antigen

 washing to remove unbound molecules

 addition of second antibody which will be bind to the first antibody This

antibody is linked to an enzyme

 washing to remove unbound molecules

 addition of a colorless substrate → color product → positive result

Exercise No3

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter33/animation_quiz_1.html

Competitive ELISA to detect antigen

Competitive ELISA to detect antigen

Competitive ELISA to detect antibody

Competitive ELISA to detect antibody

Antigen vs Immunogen

→ an immunogen must be an antigen, but an antigen is not necessarily an immunogen

Antigen vs Immunogen

→ DNP (dinitrophenol) = Hapten

BSA = carrier

Antibody molecular structure

 CDRs variable portions of the protein, both H and L

 Fc elicits immunological responses after Ag-Ab binding

 Complement cascade

Polyclonal antibody

 Recognize multiple epitopes on any one antigen Serum obtained will

contain a heterogeneous complex mixture of antibodies of different affinity

 Polyclonals are made up mainly of IgG subclass

 Peptide immunogens are often used to generate polyclonal antibodies that target unique epitopes, especially for protein families of high homology

Monoclonal antibody

 Detect only one epitope on the antigen.

 They will consist of only one antibody subtype Where a

secondary antibody is required for detection, an antibody against the correct subclass should be chosen.

Monoclonal antibody

Production of polyclonal antibody

 Inexpensive to produce

 Technology and skills required for production low

 Production time scale is short

 Polyclonal antibodies are not useful for probing specific domains of antigen because polyclonal antiserum will

usually recognize many domains

Production of polyclonal antibody

General advantages; Polyclonals will recognize multiple epitopes

on any one antigen which has the following advantages:

 Amplify signal from target protein with low expression level, as the target protein will bind more than one antibody molecule on the

multiple epitopes This would not be an advantage for quantification experiments as the results would become inaccurate.

 More tolerant of minor changes in the antigen, e.g., polymorphism, heterogeneity of glycosylation, or slight denaturation, than

monoclonal (homogenous) antibodies.

 Polyclonal antibodies are often the preferred choice for detection

of denatured proteins.

 Multiple epitopes generally provide more robust detection.

Production of polyclonal antibody

Disadvantages:

Prone to batch to batch variability.

 They produce large amounts of non-specific antibodies which can sometimes give background signal in some applications.

 Multiple epitopes make it important to check immunogen

sequence for any cross-reactivity.

 Risk of pathogenic contamination

Production of monoclonal antibody

Production of monoclonal antibody

 High technology required

Training is required for the technology used

Time scale is long for hybridomas

Production of monoclonal antibody

Advantages:

 Once hybridomas are made it is a constant and renewable source and all

batches will be identical – useful for consistency and standardization of

experimental procedures and results

Monoclonals detect one epitope only on any one antigen which has the

Production of monoclonal antibody

Lateral flow immunoassay

(immunochromatography)

Principe similar to ELISA (without washing steps)

→ simple to use, quick (15 min), no equipment needed

→ suitable for site analysis

on-NUCLEIC ACID-BASED METHODS

 Bacterial and viral pathogens may be pathogenic because

of the presence of specific genes or sets of genes.

 Resistance to antibiotic often are due to mutations or

presence of particular gene or genes.

→ These genes (DNA) can be used as diagnostic tools

Base paring rules

Nucleic acid-based diagnostic system

 Nucleic acid amplification techniques (PCR, LAMP…)

 DNA hybridization

 DNA fingerprinting…

PCR (Polymerase Chain Reaction)

DNA polymerase DNA dependent

 Copies DNA into DNA → DNA replication

 adds dNTP to a 3’ OH end of an existing strand (base pairing)

 DNA template (RNA)

 heat stable DNA polymerase (Taq pol)

dNTP

 primers

PCR

 PCR uses 2 sequence specific oligonucleotide

primers to amplify the target DNA.

 The presence of the appropriate amplified size

fragment confirms the presence of the target.

 Specific primers are now available for the detection of

many pathogens including bacteria (E coli, M

tuberculosis), viruses (HIV) and fungi.

While PCR is a very powerful technique, it is not possible

to achieve optimum results without optimizing the protocol.Critical parameters:

 Primer design

 Annealing temperature

 Type of DNA polymerases

 Concentration of Mg2+

 primer length is proportional to annealing efficiency: in

general, the longer the primer, the more inefficient the

annealing

 the primers should not be too short as specificity decreases

 Melting temperature (Tm):

 the goal should be to design a primer with an annealing temperature > 50°C

 the relationship between annealing temperature and melting temperature is one of the “Black Boxes” of PCR

 a general rule-of-thumb is to use an annealing temperature that is 5°C lower than the melting temperature

 the melting temperatures of oligos are most accurately calculated using nearest neighbor thermodynamic calculations: Tm = H [S+ R ln (c/4)] –273.15 °C + 16.6 log

10 [K+] (H is the enthalpy, S is the entropy for helix formation, R is the molar gas constant and c is the concentration of primer)

• both of the primers should be designed such that they have similar melting

temperatures If primers are mismatched in terms of Tm, amplification will be less efficient or may not work: the primer with the higher Tm will mis-prime at lower

temperatures; the primer with the lower Tm may not work at higher temperatures.

 primer specificity:

 Primer specificity is at least partly dependent on primer

length: there are many more unique 24 base oligos than there are 15 base pair oligos

 Probability that a sequence of length n will occur randomly

in a sequence of length m is: P = (m – n +1) x (¼)n

 complementary primer sequences:

 primers need to be designed with absolutely no intra-primer homology beyond 3 base pairs If a primer has such a region

of self-homology, “snap back” can occur

 another related danger is inter-primer homology: partial

homology in the middle regions of two primers can interfere with hybridization If the homology should occur at the 3' end

of either primer, primer dimer formation will occur

PCR end point analysis

 Conventional PCR is typically analysed by electrophoresis and visualisation of the amplicon (PCR product) on an agarose gel Visualisation is achieved through the use of a fluorescent dye such as ethidium bromide…

 This occurs at the end of the PCR reaction → end-point

analysis.

Real-time PCR

Three distinct phases during a PCR reaction

Real-time PCR

 Exponential phase – exact doubling of product every

cycle (assuming 100% efficiency) → very specific and precise

 Linear phase – highly variable, reaction components

starting to be consumed, and the reaction is slowing The extent of slowing will vary from replicate to replicate

 Plateau/end-point – the reaction has stopped, and no more products are being prepared Final yield will vary significantly between replicates

How does it work, real-time PCR?

Two commonly used approaches:

 Double stranded DNA detection

 TaqMan method

How does it work, real-time PCR?

Double stranded DNA detection:

 Use a fluorescent dye which specifically binds to double stranded DNA (intercalating agent, e.g SYBR green)

 PCR proceeds as normal, and the dye intercalates into the double stranded amplicon The more amplicon is

produced, the more dye is intercalated → the more

fluorescence

How does it work, real-time PCR?

TaqMan method (5’ nuclease):

 Taq pol has a 5’-3’ exonuclease activity → hydrolyses DNA on the same strand as the newly synthesised DNA

 Oligonucleotide probe contains 2 functional groups: a 5’ fluorophore, and a 3’ or quencher Energy generated by the excitation of the 5’ fluorophore is captured by the 3’ quencher → no fluorescence.

 The probe anneals to the target region specifically As the Taq pol

synthesises DNA, it hydrolyses the probe → cleavage of the 5’

fluorophore → emission of fluorescence, which can be detected.

 The level of fluorescence detected is proportional to amount of probe hydrolysis, and therefore the amount of amplicon synthesised

→ Suitable for quantitative analysis of a specific DNA sequence

How does it work, real-time PCR?

Fluorescence threshold

How does it work, real-time PCR?

 Real time PCR data is presented as CT (Cycle

threshold) values, defined as the thermal cycle at

which the fluorescence reaches an arbitrary threshold

 Use standards with known concentrations of initial

template DNA → generate a linear plot of CT vs log [initial template]

 This plot permits linear regression analysis, allowing the calculation of the copy number of any unknown target relative to the standards

How does it work, real-time PCR?

Advantages Real-time PCR

 The most accurate & feasible technique to determine the amount & concentration of

products.

 Rapid cycling (30 minutes to 2 hours).

 Specific & sensitive.

Real-time RT-PCR for diagnosis of HIV

 HIV has a ssRNA genome → use RT-PCR (reverse transcriptase

PCR).

 Lyse plasma cells from the potentially infected person to release HIV RNA genome.

 The RNA is precipitated using isopropanol.

 Reverse transcriptase is used to make a cDNA copy of the RNA of

the virus which will be used as a template in PCR reaction

 Specific primers are used to amplify a 156 bp portion of the HIV gag

gene

 Using standards the amount of PCR product can be used to

determine the viral load → determine the effectiveness antiviral

therapy.

Real-time RT-PCR for diagnosis of HIV

NASBA (Nucleic acid sequence

based amplification)

 Use 2 different primers specifically designed to recognize

2 distinct regions on the target gene

 Enzyme mixture: avian myeloblastosis virus (AMV),

reverse transcriptase (RT), T7 RNA polymerase and

RNase H

 Incubate at a constant temperature (41-42°C), 90-120 min

 Used for RNA amplification (DNA: need of denaturation step)

→ Simple, rapid, specific and sensitive technique for

amplification of target gene

NASBA (Nucleic acid sequence based amplification)

NASBA (Nucleic acid sequence based amplification)

LAMP (Loop-mediated isothermal

amplification of DNA)

 Use 4 different primers specifically designed to recognize

6 distinct regions on the target gene

 DNA polymerase with strand displacement activity

 Incubate at a constant temperature (60-65°C)

 High amplification efficiency (109-1010 times in 15-60

minutes)

 Amplification can be done with RNA templates simply

through the addition of AMV reverse transcriptase

→ Simple, rapid, specific and sensitive technique for

amplification of target gene

LAMP (Loop-mediated isothermal amplification of DNA)

http://loopamp.eiken.co.jp/e/lamp/anim.html

LAMP (Loop-mediated isothermal amplification of DNA)

LAMP (Loop-mediated isothermal amplification of DNA)

LAMP (Loop-mediated isothermal amplification of DNA)

+ Mg 2+

White precipitate Mg2P207

Visual detection of LAMP product

LAMP (Loop-mediated isothermal amplification of DNA)

Visual detection of LAMP product

DNA hybridization

For DNA hybridization:

 A probe is needed which will anneal to the target nucleic acid.

 Attach the probe to a solid matrix e.g membrane.

 Denaturation of both the probe and target.

 Add the denatured target in a solution to the probe.

 If there is sequence homology between the target and the

probe, the target will hybridize or anneal to the probe

 Detection of the hybridized complex e.g by autoradiography,

chemiluminescence or colorimetric.