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Veterinary Virology DMP851 > Viral DIagnostics: Diagnosis of disease > Flashcards

Viral DIagnostics: Diagnosis of disease Flashcards

1
Q

In Direct antibody test

A

uses primary and secondary antibodies

antibody tests can be used for testing of patient’s serum for specific viral antibodies

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2
Q

Enzyme linked Immunosorbant Assay

A
  • indicator antibodies bonded to enzymes which catalyze a reaction yeilding a visible end product
  • The product can be observed visibly or autonated with a spectrophotometric reading
  • ELISA is a very sensitive assay
  • The assay is easily automated
  • can be used to quantitate antigen or antibody
  • Many different types of ELISA
    • indirect / direct
    • Sandwich / capture
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3
Q

Virus Neutralization tests: Serum neutralization tests

A
  • Used to identify antibodies in patient serum that neutralize viruses
  • The living cell systems are inoculated with a mixture of serum which will be tested, and with virus
  • In the presence of neutralizing antibodies, virus recepotors are blocked by these antibodies, and the expected effects have not been produced
  • NO INFECTION
    • no cytopathic effect in cell cultures if virus capable of CPE
    • No lesion production in embryonated eggs
    • No clinical symptom or death in animals
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4
Q

Serum/Virus Neutralization Assay

A

requires tissue culture or animal host

Not all labs perform neutraliation assays

most informative serological assay about “protective antibodies”

Important tool used to characterize “serotypes”

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5
Q

Virus Neutralization Assay

A
  • Turn around time 6-14 days
  • Virus specific antibodies will bind to the virus and block virus entry into the cells
  • In viruses that cause cytopathic effect these antibodies will block this effect
  • Virus type specific assay and interpretation should be done with caution
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6
Q

Example Confirmatory assay - Western Blot

A
  • Infectious agent specific proteins transferred to nitrocellulose membrane
  • IgG or IgM antibodies binds to antigens on strip
  • Conjugated anti-IgM added to strip
  • Substrate converted to purple band number and/or intensity of reaction determines positive or negative results
  • Detects IgM antibody produced against infectious agent
  • Highly specific and sensitive
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7
Q

Hemagglutination Inhibition

A

serum antibodies to virus

interfere with agglutination

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8
Q

Agar Gel Immunodiffusion Assay (AGID)

A
  • Assay is easy to perform and does not require complex equipment in the laboratory
  • Assay can be set-up in many labs if reagents are available
  • Used as reference test for many disease
  • Principle of double diffusion:
    • serum is placed in one well
    • Ag placed in adjacent well
    • Antibody and Antigen diffuse thru the agar, and form a line
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9
Q

Specific Diagnostic Approaches:

Genotypic Detection Method

A

genotype = the entire genetic make-up of a virus

Looking for the presence of a specific nucleic acid sequence

Genomic nucleic acids or transcriptional RNA

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10
Q

Polymerase Chain Reaction - PCR

A
  • Rapid amplification of a DNA fragment
  • Exponential amplification
    • 2^30 > 1 billion
    • 2^35> 34 billion
    • 2^40 > 1 trillion
  • End-point visualization
  • cloning
  • Sequencing
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11
Q

Real-time PCR detection chemistry

A

SYBR green

Taqman probe

molecular beacon

Scorpion primers

Plexor

Cycling probe

Light upon eXtension (LUX)

Amplifluor / sunrise

Locked Nucleic acid (LNA)

Other binding enhancement methods

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12
Q

Taqman probe

A
  • Exploits Taq polymerase’s 5’ exonuclease activity
  • 5’ end has flurophore reporter, with a quencher molecule nearby
  • Probe is specific to gene sequence – will not fluoresce without amplification
  • Thermal cycles of PCR reaction
    • denaturation
    • Annealing
    • Elongation
    • Sometimes cycles could be combined
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13
Q

Why real-time PCR

A

Quantification

Sensitivity

Accuracy

Repeatability

analyze dozens of genes at a time

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14
Q

Real-time PCR data acquisition-CT

A
  • Camera measures the fluorescence of the well following each extension step
  • The fluorescence is plotted on a graph with realtive fluorescence on the y-axis and cycle number on the x-axis
  • During data analysis, the threshold cycle is determined and used to calculate gene expression levels
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15
Q

Recent Emerging Non FAD swine threats

A

Genotype identification and sequene determination are valuable tools for monitoring strains in herds

PRRSV

PCV2d, PCV3, PCV4

APPV

Influenza A virus

Seneca Valley Virus

Tescho Viruses

PEDV
Porcine Deltacoronavirus

Other Disease

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16
Q

“bug hunting” new tools of the trade

A
  • Non Biased Diagnostics – deep sequencing 454 Pyrosequencing
    • MiSeq
    • Ion torrent
  • Micro array
  • Proteomics
    • Mass Spec
      • MALDI-TOF
  • Genome Sequence – molecular signature
17
Q

Sample Type – From Oral Fluids

A
  • Bocavirus, parvovirus, sapovirus, picobirnavirus, various small circular DNA viruses, Kobuvirus, astrovirus, enterovirus, rotavirus, adeno-associated virus, porcine adenovirus 5, orthoreoviurs, posavirus and porcine parainfluenza virus 1.

Nasal swabs are suitable specimens for viral surveys using metagenomics

18
Q

Sample Type – from Nasal Swabs

A
  • Bocavirus
  • hemagglutinating
  • encephalomyelitis virus
  • Parvovirus
  • Porcine cytomegalovirus
  • Sapelovirus
  • Transmissible gastroenteritis virus
  • Picobirnavirus
  • Various small circular DNA viruses
  • Kobuvirus
  • Astrovirus
  • Enterovirus
  • Rotavirus
  • adeno-associated virus
19
Q

From Tissues or Serum

A

PCV

Pesti

PPV

Posavirus

20
Q

New or Never Formally identified in the US

A

Porcine parvovirus 6

Porcine pestivirus 1

Posavirus

Porcine parainfluenza virus 1

Porcine enterovirus G

21
Q

Use of Sequencing in Diagnostics

A
  • Target (contians NA of pathogen)
    • tissue
    • swab
    • blood
    • hair follicle
    • Oral fluid
    • Meat
    • juice
  • Extract NA and generate PCR product of a unique region of the genome via specific primers
  • Generate and Align sequences
  • Analysis and dendogram
22
Q

Evaluation / Verification / Validation

Why?

A

Evaluation investigates the assay performance

Validation defines the performance characteristics

Verification checks the performance characteristics

Sensitivity

Specificity

Accuracy

Percision

23
Q

Validation of assay

A

diagnostic sensitivity and Specificity

24
Q

To remember: Choose Appropriate Assays

A
  • Identify goals of testing
  • Know your clients
  • Use more than one assay to answer questions / goals
  • Test convalescent serum when possible
  • Know pathogenesis of animal diseases
  • Understand assay that you are using and your client’s needs
  • Have quality data to build confidence in your decisions
  • If you still have questions call the Diagnostic lab to ask questions associated with case

Veterinary Virology DMP851 (12 decks)

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