Molecular Epidemiology

Question 1

What is molecular epidemiology?

Answer 1

Application of techniques of molecular biology to epidemiology (i.e. study of distribution/determinants of disease, and the application of this study to control of diseases and other health problems)

Question 2

The four aspects of molecular epidemiology.

Answer 2

Identification and diagnosis, resistance/susceptibility, phylogenetic relationships, population structure.

Question 3

Identification and diagnosis

Answer 3

Detection and characterisation of infections

Monitoring parasites in the environment

Question 4

Resistance/susceptibility

Answer 4

Molecular basis of resistance

Mapping spread of resistance alleles

Question 5

Phylogenetic relationship

Answer 5

Origin and dispersal of pathogens

Discrimination between species/strains

Question 6

Population structure

Answer 6

Levels of gene flow between populations

Predicting spread of heritable traits

Question 7

Why target organelle DNA/amino acid sequences?

Answer 7

High copy number within a cell
Many complete genomes known
Design of universal primers

Question 8

Why target nuclear DNA sequences?

Answer 8

Highly repetitive elements or single copy genes

DNA sequences have different evolutionary dynamics

Question 9

What is the purpose of real-time PCR?

Answer 9

An assay that monitors accumulation of a DNA product from a PCR reaction in real time. Can quantify the amount of DNA in a sample at the start of a reaction. Increasing fluorescence as band amplified in real time.

Question 10

Real-time PCR process

Answer 10

An oligonucleotide probe is added to the reaction (fluorescent reporter and quencher, the quencher reduced fluorescence intensity)
During PCR the probe denatures and anneals to the target sequence.
For every amplification, a fluorescence probe is released following complete polymerisation of DNA strand by Taq

Question 11

Three primary steps of PCR

Answer 11

Denaturing, annealing and elongating

Question 12

What is LAMP

Answer 12

Loop-mediated isothermal amplification. A target sequence is amplified with 2/3 sets of primers and a polymerase with high strand displacement activity (as well as replication activity)

Question 13

Advantages of LAMP over PCR

Answer 13

Constant temperature. No thermal cycler. Higher amount of DNA produced (nature of loop primers). Can conduct in field.

Question 14

How is DNA detected in LAMP?

Answer 14

Turbidity - (by photometry) by increased levels of magnesium pyrophosphate precipitate
Intercalation or direct labelling of molecule - DNA-binding dyes (SYBR green (naked eye)/SYTO 9 (fluorescence))

Question 15

What are isoenzymes?

Answer 15

Enzymes which differ in their amino acid sequence but catalyse the same reaction.

Question 16

How are isoenzymes analysed?

Answer 16

Protein from the DNA separated by electrophoresis. Use dyes which respond to enzyme activity to identify. Electrophoresis will run at different time lengths depending on the species.

Question 17

AFLP process.

Answer 17

Amplified fragment length polymorphism.
Genomic DNA extract is digested with a restriction enzyme (EcoR1, BamH1). Adapters are ligated to the end of restriction fragments and PCR amplifies using primers complimentary to the adaptor. Bands are visualised either on a agarose or capillary gel electrophoresis.

Question 18

What does AFLP show?

Answer 18

Genetic variation across a whole genome

Question 19

Microsatellite analysis process.

Answer 19

The genomic DNA extract is run through PCR to amplify the microsatellite loci using fluorescently labelled 5’ primer. Capillary electrophoresis is used to determine fragment sizes NOT GELS

Question 20

What does microsatellite analysis show?

Answer 20

Repetitive regions in genome that can vary In size. These regions are non-coding.

Question 21

What does SNP analyse?

Answer 21

Single nucleotide polymorphisms look for one base change. It may be associated with drug resistance.

Question 22

Expensive detection techniques

Answer 22

PCR and sequencing, NGS sequencing

Question 23

Moderately priced techniques

Answer 23

Real-time PCR, SNP microarray

Question 24

Cheap detection techniques

Answer 24

RFLP and PCR-RFLP

Question 25

What is PCR-RFLP?

Answer 25

PCR-Restriction fragment length polymorphism is a technique which amplifies with specific primers. It digests fragments with restriction enzymes which may cut within fragment.

Question 26

How can real-time PCR detect SNPs?

Answer 26

Melt curve analysis - PCR amplify target in presence of Syber green which binds to dsDNA. Different sequences melt at different temperatures
Fluorescently-labelled probes

Question 27

SNP microarray process.

Answer 27

Good for broad coverage.
Genomic DNA/RE Digestion/Adapter ligation/PCR one primer amplification and complexity reduction/fragmentation and labelling/hybridization and wash

Question 28

Next generation sequencing

Answer 28

Whole genome or portion of genome is digested into small fragments. Fragments are sequenced and aligned to reference genome

Question 29

What drugs are P.falciparum becoming resistance to?

Answer 29

Chloroquine and sulphadozine-pyimethamine (Fansidar)

Question 30

Drugs and SNPs associated with resistance.

Answer 30

Chloroquine (chloroquine resistance transporter pfcrt 76T, multidrug resistance transporter pfmdr1)
Lumefantrine (pfcrt and pfmdr1 mutaions (and increased pfmdr1 copy number)
Amodiaquine (pfcrt and pfmdr1 mutations)
Sulphadoxine pyrimethamine (SP) dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps)
Artemisinin (number of candidates)

Question 31

What is the WWARN?

Answer 31

Worldwide antimalarial resistance network - locates resistance markers worldwide

Question 32

What did Ix of Ascaris and Trichuris zoonotic potential show?

Answer 32

Ascaris in humans and pigs morphologically similar.
In Denmark maybe similarities as hygiene better therefore those exposed probably work with pigs. Same worm, cross transmission.

Question 33

Five ebola species

Answer 33

Zaire ebolavirus 
Sudan ebolavirus
Reston ebolavirus
Tai Forest ebolavirus
Bundibugyo ebolavirus