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Bacteriology/ Mycology > Genetics, Vaccines, Diagnostics > Flashcards

Genetics, Vaccines, Diagnostics Flashcards

1
Q

What is a tranformant?

A

• Transformant- cell that has taken DNA by bacteria

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2
Q

What is a competent?

A

• Competent- bacteria that can take up DNA naturally (i.e.
without artificial treatment)

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3
Q

What is Homologous recombination?

A

• Homologous recombination- identical or similar regions of
DNA align and exchange each other by excision-repair
– also known as allelic exchange or strand replacement (occurs a-lot in nature)

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4
Q

What is transfection?

A

• Transfection- uptake of viral DNA

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5
Q

What is the mechanism of transformation?

A

• DNA present from lysed cell, most will be degraded, but some may be taken up by another cell nearby. If this DNA offers that cell an advantage it will be replicated and incorporated as the cells reproduce.

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6
Q

Why is transformation important?

A
  • If DNA was material responsible it can provide explanation to how bacteria can acquire other genes.
  • Provides basis of the development of artificial transformation schemes ( electroporation)
  • Allows the easy production of mutants to allow for the definition of gene function ( virulence determinants)
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7
Q

What is artificial transformation schemes?

A

introduce “foreign genes” into both eukaryotic and prokaryotic
cells. - allowed for the easy production of mutants to allow for
the definition of gene function e.g. virulence determinants.

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8
Q

What is seen in this image?

A

Bacteriophage

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9
Q

What are these dark circles representing in this image?

A

• Dark circles represent areas of Salmonella growth lysed by phage

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10
Q

What is the mechanism of transduction?

A
  • Virus infects cell -> Takes up cells machinery and will replicate itself. -> Virus will make more of itself and sometimes take up some of the bacterial DNA -> When those viral cells infect other cells, they will also transfer some of that bacterial DNA into the cell -> if this DNA offers that cell an advantage, it will be replicated for future generations. It will eventually outgrow the resident population
  • This happens alot
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11
Q

What would be the reason that bacteria would keep the new DNA?

A

It has to provide the cell an advantage.

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12
Q

What occurs in a lysogenic phase of transduction?

A

bacteria with new DNA gene becomes part of the cell and is inactivated. Can be reactivated later

• Chicken pox -> Shingles ( DNA remains part of that host cell for life and can be reactivated)

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13
Q

What occurs in the lytic phase of transduction?

A

Bacterial particles are replicated in cell and cell lyses releasing contents to infect more cells.

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14
Q

What casued the O antigen in salmonella?

A

• O antigens in salmonella have been brought in by bacteriophages.

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15
Q

What are the 3 extrachromosomal pieces of dna?

A

◦ Plasmids
◦ F- Factor ( via sex pillus)
◦ R- Factor

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16
Q

What is a plasmid?

A

Plasmids = genes of unknown origin and often not
necessary for growth; others contain genes encoding
virulence factors

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17
Q

What is an F Factor?

A

2) F-factor = fertility factor; genes promote replication
and transfer of the factor to recipient cells e.g. conjugation.

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18
Q

What is an R factor?

A

3) R-factor = F factor + genes encoding resistance to
specific antibiotics.

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19
Q

What is conjugation?

A
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20
Q

What are the 2 genes inside of an R factor?

A

◦ 1) Resistance transfer factor = RTF encodes origin of replication, sex pilius genes.
◦ 2) r-determinants = genes encoding antibiotic resistance, heavy metal resistance or other virulence factors (determinants), insertion sequences (IS)

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21
Q

What is transposons? How can they jump? What sites does this occur on? What will allow it to replicate?

A

Transposons- or insertion sequences/ jumping genes
• can jump between one region of chromosome to another, in and out of plasmid, ect.
• They do this due to sites called an IS regions ( insertion sequences)
• Transposons tend to go into certain regions more commonly in the gene.
• It only replicates if it can provide a benefit to that cell.

22
Q

Are bacteria intelligent?

A

• Nope just replicate faster than us and they mutate faster, so we need to find ways to try to be 3 steps ahead. .

23
Q

how can you prevent antibiotic resistance?

A

• Way to combat antibiotic resistance, is to not use the antibiotic.

24
Q

What is a live attenuated vaccine?What are the pros and cons of live attenuated vaccines?

A

• Live: attenuated - limited growth in host
◦ Pros: allows for immune response
◦ Cons: can return to virulence if we are not careful, can cause mild symptoms of disease.

25
Q

What is a killed vaccine?

A

• Killed vaccine: chemically inactivated whole cells. most common

26
Q

What are the limitations of a live attenuated vaccine?

A

Live – attenuated - reversion to disease causing status ?

27
Q

What are the limitations of a killed vaccine?

A

• Killed - not very effective; too many antigens, and tend to stimulate only a short-lived Th2 response

28
Q

What are the limitations of a subunit vaccine?

A

Subunit - useful but often expensive & need to be mixed with adjuvants to be effective

29
Q

What are the limitations of a live recombinant vaccine?

A

Live recombinant - regulatory hurdles & public negative to use of recombinants

30
Q

What are the limitations of a DNA vaccine?

A

• DNA - still needs refinement and way to avoid gene silencing by host

31
Q

What is the goal of putting a gene into a high copy plasmid?

A

Put a gene into a high copy plasmid and you make a-lot of antibodies. This could be a way to use attenuated vaccines and create an immune response safely.

32
Q

What is one way to make a vaccine?

A

Change an already made vaccine

33
Q

What made the vaccine for brucella? Who cannot receive this vaccine?

A

The vaccine for brucella was made when the bacteria it lost its O antigen and it was not virulent unless the animal is pregnant ( can not be given to pregnant animals)

34
Q

How were they able to vaccinate for anthrax?

A

• Can take this protective PAG gene and put it into the vaccine already made for brucella and can be used to protect against 2 diseases.

35
Q

What is PAG ? What does it encode for?

A

PAg is a gene that codes for PA. PA is a part of anthrax toxin that antibodies neutralize.

36
Q

What are the advantages of using a DNA based diagnostic test?

A

• High sensitivity
- Can theoretically detect the presence of a
single organism • High specificity
- Can detect specific genotypes - Can determine drug resistance - Can predict/confirm virulence
• Speed
- Quicker than traditional culturing for
certain organisms

37
Q

When can you use a DNA test?

A
  • Culture to identify/ confirm identification
  • Low #s or difficult to isolate/ grow (i.e mycobacterium, fungi, viruses)
  • Organisms present in small volume specimen (i.e ocular fluid, forensic samples)
  • Molecular epidemiology
  • To identify point sources for hospital and
    community-based outbreaks
  • Zoonotic/ highly infectious agents to minimize hazards.
  • Non viable organism (tied up in immune complex)
38
Q

What are the disadvantages of DNA diagnostic testing?

A

Disadvantages is DNA must be present for it to work.
Expensive

Can be problematic for mixed culture infections.

39
Q

What is PCR? Is it a diagnostic test?

A

It is not a diagnostic test but an amplification tool.
Can amplify the DNA even if its not viable.
• can detect a really small quantity of DNA

40
Q

Why was PCR very manual previously? What has changed that has allowed it to be more automated?

A
  • Used to be very manual because when the Denaturation would take place at 94 degrees Celsius the DNA polymerase would be destroyed.
  • They now use a Thermophile DNA polymerase it can withstand those temperatures and it is a lot more automated.
41
Q

What causes brucellosis?

A

Causative agent: gram negative,
cocco-bacillus, genus Brucella.

42
Q

What does brucellosis cause?

A

Disease: brucellosis = abortion in livestock
caused by Brucella abortus

43
Q

What diagnostics can be used to diagnose the condition?

A

Diagnosis: indirect - serum serology, antibodies to O-side chain
direct – culture, identification

44
Q

What is the isssue with brucellosis which make it difficult to test for?

A
  • all 8 Brucella species grow very slow, 3-7 days
  • biochemical phenotyping requires multiple tests - vaccine strain RB51can cause abortion IF
    given to pregnant cow!
45
Q

What is a real time PCR?

A

• Real time PCR is very good for diagnostics and is very sensitive.

46
Q

How does Real Time PCR work ( TAC band)

A

• TAC band
• you have a reporter and a quencher. The reporter is fluorescent and the quencher inhibits it. When they are on the same DNA the quencher inhibits the fluorescence. Once it amplifies a specific
piece of DNA, the DNA polymerase is a 5 prime nuclease and it chews up the probe and releases the reporter from the quencher. Now the reporter fluoresces and will give off a signal, but only if the DNA was amplified and the probe/primer was very specific to that section of DNA

47
Q

What can be indicated by the number of cycles it takes for a result in a real time PCR?

A

• The number of cycles it takes gives you an idea of how much DNA was there

48
Q

What are the advantages of Real Time PCR?

A

Fast, automated, can do alot of tests at once. But expensive.

49
Q

What is the best diagnostic method we have currently for most bacteria?

A

MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time Of Flight) Mass Spectrometry

50
Q

Why is MALDI-TOF system considered the best diagnostic tool out there right now?

A

• VERY SENSITIVE. Can give information down to species level. And if it is one of the ones it doesnt know which is only like .1 % of the time, it will say it cant identify it rather than give the wrong one.

51
Q

What is PCR? What is Real Time PCR? What is MALDI- TOF?

A
  • PCR- amplification not diagnostic. Real time PCR is diagnostic. IF DNA you are looking for is there it will tell you right away
  • Malditof is the fastest, most accurate, and least expensive diagnostic method available.
  • Protein is based on RNA in the sample. Which is a byproduct of the DNA.
52
Q

What is the benefit of recombinant DNA?

A

Allows for vaccine enhancement by:
- over-expression of genes relatively easy
• for purification

  • antigen presentation- subunit
  • diagnostic reagents ( ie. elisa)
  • over expression of genes in an attenuated pathogen
  • targeted gene mutations are very easy- attenuated pathogen
  • creation of DNA vaccine- stable/ non disease producing

Bacteriology/ Mycology (19 decks)

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