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Flashcards in molecular diagnostics Deck (11):
1

Diagnostic techniques (4)

Diagnostic techniques

  • direct examination
  • culture
  • immunologic systems
  • molecular analysis

 

2

specificity vs. sensativity

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**

3

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

Molecular detection and identification

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

4

Electrophoresis

Molecular Detection & Identification

Electrophoresis

= separated in an electrophoretic field

(negative molecules travle towards positive pole)

  • mobiliy based on
    • molecule size
    • structure

 

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5

RFLP

 

Molecular Detection & Identification

Restriction Fragment Legnth Polymorphism

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

 

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6

Hybridization & Probe

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)

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7

Nuclear acid amplification

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! 

8

Primers

(relation to PCR)

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

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9

PCR variations (4)

(elaborate on one)

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

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10

LAMP

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

11

Western blot

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