Systems for detection of pathogens 2

Question 1

What is molecular gene targeting ?

Answer 1

Nucleic acid amplification techniques (NAAT)
Polymerase Chain Reaction (PCR)

Aim to detect a gene or gene products that are pathogen specific

Question 2

Outline PCR

Answer 2

Polymerase chain reaction

Two DNA primers , specific for opposite DNA strands used to amplify DNA region

Product is visualised by fluorescent tags or staining in gels for an amplicon of an exact size

From one copy to many copies
Because there are many copies, the genes can now be easily seen compared to the original one copy

Question 3

What is QuantitativePCR ?

Answer 3

This measures the speed at which a PCR amplicon product accumulates by the amount of fluorescence released.

Much more reliable

Question 4

What is SDA ?

Answer 4

This is strand displacement amplification.

It is a similar process to PCR

Question 5

What are the factors which determine if a molecular test is good enough ?

Answer 5

CHECK SLIDE

Question 6

What are microarrays ?

Answer 6

These are multiple gene targeting

Ordered short oligonucleotides probes attached to slides in defined spots

Each spot represents a single gene

Comparative Genomic Hybridisation (CGH) DNA

Very small slide so a small sample can be used

Question 7

What are the advantages of microarrays ?

Answer 7

It can be used to look at whole genome
Strand dependent 
Can be used for RNA and Transcriptomics 
Can look for microRNA 
The RNA can show what the organism is expressing

Question 8

What is Mass spectrometry ?

Answer 8

Isolate organism
Lyse with crystallising matrix
Ionise and detect time of flight for each particle
There is a detector at the end

Peaks are formed small first and larger at the end

Mwt (Daltons) for each produced protein can then be calculated

The produced patterns can be compared againsta database to identify the microorganism

Question 9

What are the advantages and disadvantages of MALDI TOF profiling ?

Answer 9

SLIDE

Question 10

What are the biomarkers of virulence ?

Answer 10

Virulence is the severity or harmfulness of a disease

Gram negative cell wall has many polysaccharides and this is what antibodies are created against

Question 11

What is the latex agglutination test ?

Answer 11

This is a test which uses particle coated with specific antibody to cell wall antigens. The antibody will cause the beads to agglutinate.
Very specific test

Question 12

What is serotyping ?

Answer 12

a distinct variation within a species of bacteria or virus or among immune cells of different individuals. These microorganisms, viruses, or cells are classified together based on their cell surface antigens

Question 13

How can we serotype ?

Answer 13

Using :
CSF direct agglutination test
Serology by ELISA

TOXIN DETECTION

Question 14

What are the advantages and disadvantages of Biomarkers of virulence ?

Answer 14

SLIDE

Question 15

What sort of things are being checked through sequencing in terms of virulence ?

Answer 15

To check whether the change in sequence /mutation is involved in virulence.
Silent mutations can be :
Intragenic (between genes)

Synonymous (not altering coding)

Question 16

What are the advantages of molecular detection methods?

Answer 16

SLIDE

Question 17

What are the disdvantages of molecular detection methods?

Answer 17

SLIDE

Question 18

What is a transcriptomic profile ?

Answer 18

The study of transcriptomics, (which includes expression profiling, splice variant analysis etc), examines the expression level of RNAs in a given cell population, often focusing on mRNA

Question 19

What is metabolic profiling and what way can we test this?

Answer 19

Breath test,urine test

These can be studied using nuclear magnetic resonance , gas chromotography

the measurement in biological systems of the complement of low-molecular-weight metabolites and their intermediates that reflects the dynamic response to genetic modification and physiological, pathophysiological, and/or developmental stimuli.