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House Microbiology > Laboratory diagnosis of viral infection > Flashcards

Laboratory diagnosis of viral infection Flashcards

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1
Q

Virus isolation/culture

3 types

_____
_____
_____

A

Cell culture
Egg inoculation
Animal inoculation

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2
Q

Egg inoculation

________ are among the most useful form of living animal tissue

Used for the ____ of viruses or _____ viruses

Used for _________ cultivation in the production of viral vaccines

A

Embryonated eggs

isolation; titrating

large quantity

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3
Q

Animal inoculation

In 1909, Landsteiner & Popper used ______ to isolate Polio virus.

_____,______,_______,_______ are now commonly used

A

monkeys

Rabbit, Guinea pig, mouse, rat

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4
Q

Earliest method for cultivation of viruses causing human disease is by ______

A

Animal inoculation

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5
Q

Animal inoculation

The growth of virus in inoculated animals may be indicated by _____,_____ or _______

A

death, disease or visible lesions.

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6
Q

Disadvantages of animal inoculation are that they may ______ with viral growth and the animals often _________

A

interfere

harbour latent viruses.

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7
Q

Chorioallantoic membrane inoculation is used for which viruses

A

Herpes simplex
Pox
Rous sarcoma

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8
Q

Amniotic inoculation is used for which viruses

A

Influenza
Mumps

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9
Q

Yolk sac inoculation is used for which viruses

A

Herpes simplex

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10
Q

Allantoic inoculation is used for which viruses

A

Influenza
Mumps
Newcastle disease virus
Avian adenovirus

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11
Q

Cell culture

Primary cell culture

It consists of ____ cells, freshly taken from body & cultured.

They are capable of _____ growth in culture & cannot be maintained in _____

Eg. Monkey, kidney, human embryonic kidney, human amnion & chick embryo cell culture.

A

normal; limited; serial culture.

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12
Q

Cell culture

Primary cell culture

They are useful for __________ and for ________

A

isolation of viruses & for vaccine production.

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13
Q

_______ is best cell culture systems available

Why?

A

Primary cell culture

since they support the widest range of viruses

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14
Q

Primary cell culture is quite cheap

In Primary cell culture it is often difficult to obtain a reliable supply

T/F

A

F, they are very expensive

T

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15
Q

Cell culture

Diploid cell culture

These are used to ensure ___________

After ____ serial passages, they undergo ________.
Eg. Human _______

A

a continuous supply of cell line.

50; senescence

fibroblasts

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16
Q

Diploid cell culture are useful in viral vaccine production.

T/F

A

T

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17
Q

Cell culture

Continuous cell culture

These are single type usually derived from _____ cells that are capable of __________________________________.

Eg. _____ for rabies vaccine, ___ , HEp-2, LLC-MK2

A

cancer

continuous serial cultivation indefinitely

Verocell; Hela

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18
Q

Continuous cell culture are not useful for vaccine production.

T/F

A

F

They are

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19
Q

________ cell culture is most easy to handle

A

Continuous

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20
Q

the range of viruses supported by continuous cell culture is limitless

T/F

A

F

often limited

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21
Q

Mention the types of cell cultures

A

Primary
Diploid
Continuous

22
Q

Cell cultures

Growing virus may produce

  1. ________
  2. ________
A

Cytopathic Effect (CPE)

Haemadsorption

23
Q

Cell cultures

Growing virus may produce

  1. Cytopathic Effect (CPE) - such as the ______ of cells or ______ formation, may be specific or non-specific.
  2. Haemadsorption - cells acquire the ability to _______________
A

ballooning; syncytia

stick to mammalian red blood cells

24
Q

Cell cultures

Confirmation of the identity of the virus may be carried out using ______,__________, or __________ tests.

A

neutralization, haemadsorption-inhibition or immunofluorescence

25
Q

Syncytium formation is seen in cell culture caused by ____ ,and _____ virus

A

RSV

measles

26
Q

Problems with cell culture

_____ period (up to _____ ) required for result.

Often very poor ________ ,which depends on a large extent on the _________.

Susceptible to ___________.

A

Long; 4 weeks

sensitivity; condition of the specimen.

bacterial contamination

27
Q

Problems with cell culture

Susceptible to ________ which may be present in the specimen.

Some viruses will not grow in cell culture e.g. ________,________,________,________

A

toxic substances

Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus.

28
Q

Light microscopy

Detecting the ____________ in smear & tissue.

A

viral inclusion body

29
Q

Light microscopy

Inclusion bodies are ________ of stainable substances, usually proteins.

Can either be :
_______ inclusions or ________ inclusions

A

dense aggregates

Intra nuclear

Intra cytoplasmic

30
Q

Electron microscopy

Virus particles are detected and identified on the basis of ______

______ virus particles per ml required for visualization

______-______ magnification normally used.

A

morphology

10^6

50,000 - 60,000

31
Q

Electron microscopy

Use Vesicle Fluid for ______ or ____

Skin scrapings for _____,_____,____

Faeces for ____,____,_______,______ ,______

A

HSV VZV

papillomavirus, orf molluscum contagiosum

Rotavirus, Adenovirus Norwalk like viruses Astrovirus, Calicivirus

32
Q

Molecular methods

________

_________ reaction

_______ reaction

__________ based amplification

________ mediated amplification

A

Hybridisation

Polymerase chain

Ligase chain

Nucleic acid sequence

Transcription

33
Q

Polymerase chain reaction or PCR
is a simple laboratory technique to obtain ______ of _____ fragments even from samples containing only ____ quantities of DNA or RNA.

A

multiple copies

specific DNA

minute

34
Q

Essential components of PCR

______
________
________

______ cations

Deoxynucleoside triphosphates (dNTPs)
________ solution

A

Template DNA
Primers
Thermostable DNA polymerase enzyme

Divalent

Buffer

35
Q

Essential components of PCR

Template DNA that contains the ______ of interest to be amplified during the PCR.

A

target sequence

36
Q

Essential components of PCR

Primers- A pair of synthetic ________ (______ and ____ primers) that are complementary to the ___’ ends of each of the two strands of target DNA.

A

oligonucleotides

forward and reverse

3

37
Q

Essential components of PCR

Thermostable DNA polymerase enzyme like ____ polymerase (originally isolated from ————————- ) is a vital ingredient of a PCR to _____ the _____________________ of DNA.

A

Taq

thermophilic bacterium Thermus aquaticus

catalyse; template-dependent synthesis

38
Q

Essential components of PCR

Divalent cations, usually ____ are required in (minute or optimum?) concentration for the activity of most __________ as well as for several other steps in PCR.

A

Mg 2+

optimum

thermostable DNA polymerases

39
Q

Essential components of PCR

Deoxynucleoside triphosphates (dNTPs)-

________ amounts of each dNTP (dATP, dCTP, dGTP, dTTP), which are ______ used by the DNA polymerase enzyme to synthesize a new strand of DNA.

A

Equimolar

building blocks

40
Q

Essential components of PCR

Buffer solution to maintain ____________ for optimum activity and ______ of the _____

A

suitable ionic environment

stability

DNA polymerase.

41
Q

STEPS IN PCR

______
————
________

A

Denaturation
Annealing
Extension

42
Q

STEPS IN PCR

Denaturation: The first step of a PCR where the sample is ____ to _____ or _____ the two strands of the DNA (>_____oC).

A

heated

separate or denature

90

43
Q

STEPS IN PCR

Annealing: the reaction temperature is ______ (usually ___-__ 0C below the Tm of _____ )to allow the ————- to bind to the single strands of the template DNA.

A

lowered

3-5; primer

oligonucleotide primers

44
Q

STEPS IN PCR

Extension: the temperature is ______, typically to ____°C, allowing specific enzymes to synthesize a new DNA strand complementary to the DNA template.

A

Raised

72

45
Q

STEPS IN PCR
Typically about ___-___ cycles of PCR are performed depending upon the _________ used, the __________ and the ______________ for post-PCR processing.

A

25 to 45; type of PCR ; amount of initial template DNA ; number of amplicon copies desired

46
Q

STEPS IN PCR

The PCR is commonly performed in a reaction volume of ____ μl in small reaction tubes (___-___ ml volumes) in a _______ that heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction.

A

10–200; 0.2–0.5

thermocycler

47
Q

Immunological assay

Antigen detection

•_________ assay (ELISA)
•_____________
•___________/_________ assay

A

Enzyme linked immunosorbent

Immunoflourescence

Immunochromatographic /lateral flow

48
Q

Immunological assay

Antibody detection and quantification

•_____
•_____________ test
•__________ test

A

ELISA

Complement fixation

Immunoflourescence Rapid

49
Q

Post PCR analysis

Agarose gel electrophoresis.

PCR products can be visualized by staining the gel with _______ such as ______ which binds to DNA and intercalates between the stacked bases.

Confirmation of size of the DNA product is done by ______________________

A

fluorescent dye

ethidium bromide

comparing the size with DNA ladder.

50
Q

The appearance of discrete band of the correct size may be indicative of a successful PCR amplification.

T/F

A

T

51
Q

POST PCR analysis

______ electrophoresis

______ the PCR product

__________ with a specific _______

A

Agarose gel

Sequencing

Hybridization; oligonucleotide probe

House Microbiology (93 decks)

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