molecular diagnostics

Question 1

Diagnostic techniques (4)

Answer 1

Diagnostic techniques

• direct examination
• culture
• immunologic systems
• molecular analysis

Question 2

specificity vs. sensativity

Answer 2

both are measurments of the quality of a molecular test/identification/detection

• Specificicity= true negative rate
  
  • measures porportion of negatives that are correct
  • if a test is more specific, it will have more negatives because it clearly and accurately targets only the specific thing
• Sensativity= true positive rate
  
  • measures porportion of positives that are correct
  • if a test is really sensative, it will easily/often detect the pathogen wether there is a lot or a little present

***we aim to reduce sependency on cultures because of safetly concerns thus maximizing specificity and sensativity of tests is a goal in molecular diagnostics**

Question 3

List methods of molecular detection and identification of microorganisms (5)

Answer 3

Molecular detection and identification

• electrophoresis
• RFLP- restriction fragment length polymorphism
• hybridization & probe
• nuclear acid amplification- signal or target
• protein detection- western blot, proteomics

Question 4

Electrophoresis

Answer 4

Molecular Detection & Identification

Electrophoresis

= separated in an electrophoretic field

(negative molecules travle towards positive pole)

• mobiliy based on
  
  • molecule size
  • structure

Question 5

RFLP

Answer 5

Molecular Detection & Identification

Restriction Fragment Legnth Polymorphism

restriction enzymes- cut DNA at apecific recognition nucleotide sequences (SEQUENCE SPECIFIC)

Question 6

Hybridization & Probe

Answer 6

Molecular Detection & Identification

Hybridization

= use a “probe” which binds to complimentary single- stranded sequences

probe is a fragment of nucleic acids

• can be labled
• detects complemtary sequence (high specificity)

Question 7

Nuclear acid amplification

Answer 7

Molecular Detection & Identification

Nuclear Acid Amplification

= amplify stuff enough to easily recognize it. signal or target.

• signal amplification
  
  • bDNA assays or hybrid capture assays
• Target Amplificaiton= enzyme mediated process to synth copies of target nucleic acid
  
  • PCR- polymerase chain reaction
  • LAMP- isothermal amplification
  • ** high sensativity but false positives!

Question 8

Primers

(relation to PCR)

Answer 8

nuclear acid amplification

target amplification

primers= piece of RNA that is starting point for dna synthesis

• can be random or specific

PCR PRIMERS

• target complementary sequences that flank the region to be amplified
• distance between the primers determines legnth of product
• determine specificity

Question 9

PCR variations (4)

(elaborate on one)

Answer 9

PCR variations

• reverse-transcriptase PCR
• nested PCR
• multiplex PCR
• quantitative/real-time PCR
  
  • ​generates an exponential curve with lag phase, log phase, and exponential phase.
  • lag phase inversely proportional to amount of starting material

Question 10

LAMP

Answer 10

nucleic acid amplification

target amplification

Loop-mediated isothermal amplification

likely the future of dna amplification as it is easier and more cost effective than pcr. just as accurate

• pros: quick, as good or better than PCR, visible results
• cons: design of primer sets is complicated

Question 11

Western blot

Answer 11

Molecular Detection & Identification

Protein detection

Western Blot

= method of proteomics (study of proteins) that detects specific sequences of amino acids which can then be put in database to identify protein