Molecular Diagnosis

Question 1

What are the 4 diagnostic techniques?

Answer 1

1. Direct examination
2. Culture
3. Immunologic methods
4. Molecular analysis

Question 2

Describe molecular diagnosis.

Answer 2

-RNA, DNA & Proteins = identify pathogenic agents
-safe
-reduction in dependency on culture
-sensitivity & specificity
EX:
Electrophoresis, (RFLP) restriction fragment length polymorphism, hybridization, nuclear acid amplification (target), protein detection

Question 3

Describe electrophoresis.

Answer 3

-separated in an electrophoretic field
-negatively charged molecules -> positive end
-mobility
>size: smaller = faster
>structure: supercoiled -> linear -> nicked circles (DNA)

Question 4

Describe rotavirus strains detected by SDS-PAGE.

Answer 4

-L1&2 = long profile strains
-L3 = short profile strain
SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Question 5

Describe restriction fragment length polymorphism (RFLP).

Answer 5

-analyze diff among homologous DNA sequences by restriction enzymes
-restriction enzymes: cut DNA at the specific recognition nucleotide sequences (sequence specific)

Question 6

Describe hybridization.

Answer 6

-denatured, single stranded DNA
(probe binds to a complementary single-stranded sequences)
>fragment of nucleic acids
>labeled using radioisotope, enzyme, or chemiluminescence
>detect complementary sequences
>high degree of specificity
>various in size

Question 7

Describe the nucleic acid amplification.

Answer 7

-target amplification = enzyme-mediated process to synthesize copies of targeted nucleic acid
-PCR
>isothermal amplification such as LAMP
~highly sensitive
~false positive

2^n
N = cycles

Question 8

Describe PCR primers.

Answer 8

-single stranded DNA fragments, complementary to sequences flanking the region to be amplified
-distance between primer binding sites determines the size of the PCR product
-determines specificity

Question 9

What are the features of primers?

Answer 9

1. Types of primer
   -random
   -specific
2. Primers
   -product size
   -annealing temp
   -specificity
3. Nucleotide composition

Question 10

What are the PCR variations?

Answer 10

1. Reverse-transcriptase PCR
2. Nested PCR
3. Multiplex PCR
4. Quantitative or real time PCR

Question 11

Describe real time or quantitative PCR (qPCR).

Answer 11

-probe/dye = generate fluorescent signal from product
-signal in real time = quantification of starting material
-signal = exponential curve with lag phase, log phase, stationary phase
-lag phase = inversely proportional to amount of starting material

Question 12

Describe the pros & cons of LAMP.

Answer 12

Pros:
-no thermal cycler needed
-quick 1 hr
-sensitivity > PCR
-visible results
Cons:
-design of primer sets complicate

EX: diagnosis of T. Tenax by LAMP & PCR

Question 13

Describe protein detection.

Answer 13

-western blot
-EPM in equine