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Microbiology > Chapter 14: Vaccines, Diagnostics, Therapeutics, and Molecular Methods > Flashcards

Chapter 14: Vaccines, Diagnostics, Therapeutics, and Molecular Methods Flashcards

1
Q

Chinese used variolation to combat smallbox 100 years ago…

A

powder made from dried scabs of smallpox; practitioner blew the powder into a healthy individuals nose; resulting small boz infections tended to be milder (only 1-2% mortality rates)

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2
Q

Edward Jenner

A

1796; milkmaids were not affected by small pox epidemics; most of them contracted cowpox; jenner suspected that a prior cowpox infection was protective against small poz; tested this by purposely inoculating a boy with cowpox pus, boy contracted it but recovered quickly he was then infected with small pox and did not show symptoms

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3
Q

Vaccination

A

derived from vacca the latin word for cow

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4
Q

Louis Pasteur developed…

A

late 1800s; early version of rabies vaccine to protect humans; vaccine to protect cattle against anthrax

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5
Q

How many different infections are currently vaccine preventable?

A

at least 25

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6
Q

The Lancet Paper

A

study of just 12 people; claimed a correlation between the measles, mumps, and rubella vaccine and the development of autism; resulted in many parents declining the MMR vaccine (as well as others); 2010 Lancet retracted the study and said it was bad science; additional studies have been done to prove it false

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7
Q

A drop in childhood vaccines has led to….

A

outbreaks of vaccine preventable disease; measles show the strongest re-emergence; resulted in states mandating vaccines

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8
Q

Herd Immunity

A

occurs when a sufficient percentage has been vaccinated (varies between pathogens); protects premature babies and immun-compromised patients; most ~85% vaccination but measles and whooping cough require ~95%

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9
Q

Live Attenuated Vaccines

A

contain altered pathogens that do not cause disease but are still infectious; developed multiple ways: cultivate pathogen in cell culture so it loses its pathogenicity (like flu strain in egg), genetic manipulation

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10
Q

Pros/Cons of Live Attenuated Vaccines

A

pro- stimulate potent immunological responses that are accompanied by long lived memory (bc it is a more natural imm response); cons- could cause disease in a immune compromised host, possible mutations to a infectious form, often need to be refrigerated

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11
Q

Inactivated Vaccines

A

include whole inactivated pathogens (the entire pathogen) or whole-agent and subunit vaccines ( a part)

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12
Q

Pros/Cons of Inactivated Vaccines

A

pros- safe for immune compromised patients and stable at room temp; cons- boosters required to achieve full immunity

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13
Q

Whole Agent Vaccines

A

contain the entire pathogen; pathogen is inactivated (not able to cause infection) by heat, chemicals or radiation

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14
Q

Subunit Vaccines

A

do not include whole pathogens; consist of purfied antigens (unable to cause disease) or parts of the infectious agent; require adjuvants; include purified subunit vaccines, toxoid vaccines, and conjugate vaccines

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15
Q

Adjuvants

A

pharmacological additives that enhance the bodys natural immune resposne to an antigen

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16
Q

Purified Subunit Vaccines

A

immunogenic portion of the pathogen; can be harvested from a natural pathogen or purified from a genetically engineered expression sys (recombinant subunit vaccines)

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17
Q

Toxoid Vaccines

A

purified and inactivated toxins; ex) tetanus and diptheria of DTap and Tdap

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18
Q

Conjugate (or polysaccharide) Vaccines

A

polysaccharide antigens cojugated to a more immunogenic protein antigen (bc immune sys no like recognizing sugars and respond better to proteins); ex) meningococcal vaccines, pneumococcal vaccines, and Hib vaccines

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19
Q

mRNA Vaccines

A

purified mRNA is encased in lipids chemically compatible with the cells plasma membrane; mRNA delivered to host cells; host cells translate the mRNA to build an antigenic protein that triggers an imm response; ex) moderna and pfizer vaccines for SARS-CoV-2

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20
Q

Recombinant Vector Vaccines

A

genetic material from the pathogen is packed inside a harmless virus or bacterium and inserted into the body; ex) johnson and john COVID vaccine

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21
Q

DNA Vaccines

A

genes encoding highly immunogenic antigens are identified; target genes are placed into a plasmid; plasmid is injected into a human host; human cells take up the plasmid and transcribe and translate the genes; cells become the antigen producers; results in a humoral (antibody) and a cellular immune response (T cells); focusing on HIV and cancer

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22
Q

Pros/COns of Biochemical Tests

A

biochemical tests detect pathways (like fermentation) and do not work with viruses; pros- useful for identifying bacteria that are responsible for an infection; cons- can take more than 24 hrs to perform, pathogen must be cultureable and cant identify noncellular pathogens

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23
Q

Antibodies

A

have 2 antigen binding sites; can attach to more than one antigen; can bind antigens into a clump (agglutination)

24
Q

Agglutination Reactions can be seen when…

A

antibodies interact with cells that display multiple surface antigens or tiny synthetic beads coated with antigens

25
Q

Treponema Pallidum Particle Agglutination Assay (TPPA) Test

A

tests for symphilis; detects patient antibodies against T. pallidum

26
Q

Plaque Reduction Neutralization Test (PRNT)

A

patients serum is extracted and serially diluted; preparation of the suspected virus is added to the various tubes of diluted serum; each serum/virus mixture is added to petri plates of cultured cells and incubated

27
Q

Negative Results of PRNT

A

no neutralizing antibodies present in the patient sample; patient sample and control culture exhibit same level of infection

28
Q

Positive Results of PRNT

A

neutralizing antibodies present in the patient sample; antibodies bind to the added viruses and neutralizes them

29
Q

Enzyme Linked Immunosorbent Assays

A

ELISAs; versatile and rapid diagnositc tests; rely on- antibody-antigen interactions, reporter enxyme attached to a monoclonal detecting antibody; chemically modifies an added substrate

30
Q

Direct ELISA

A

allows for identification of antigens in a sample; solution possibly containing antigens is added to a microtiter plate wells; antigens stick to the bottom ofthe wells; detection antibodies are added and bind if the antigen is present; unbound antibodies are washed out of wells; substrate is added

31
Q

Indirect ELISA

A

requires tow antibodies: first antibody (from patient) recognizes bound antigen and second antibody is enzme-linked

32
Q

To Perform an Indirect ELISA

A

microtier plate is precoated with antigen; patients serum is added to plates; patient antibodies that recognize the antigens bind; enzyme linked detection antibody is added (binds to specific types of human antibodies); unbound detection antibodies are washed out; substrate is added; signal levels are measured

33
Q

Performing the Sandwich ELISA

A

microtiter plate is coated with capture antibody; sample possibly containing antigen is added; antigens bind the capture antibody; detection antibody is added (sandwichs the antigen between two antibodies); unbound antibodies washed out; substrate is added and signal is measured

34
Q

Immunofluorescence Microscopy

A

utilizes fluorescent tagged antibodies to recognize a specific antigen in a sample; requires a specialized fluorescent microscope

35
Q

Immunofluorescence Assays (IFAs)

A

like ELISA, detects anitgens or antibodies in a patient sample; detection antibody is linked to a fluorescent tage instead of an enzyme; eliminates the need to add substrate for the detection step

36
Q

Flow Cytometry

A

allows for enumeration of specific cells; requires fluorescence-activated cell sorter (FACS)

37
Q

To Perform Flow Cytometry

A

fluorescent-tagged antibodies are incubated with a patient blood sample; unbound antibodies are removed; sample is loaded into the FACS machine; tagged cells are counted and sorted

38
Q

Polymerase Chain Reaction (PCR)

A

sensitive enough to detect a single pathogen in a sample; creates billions of copies of a target gene in just a few hours; applications- facilitates gene sequencing for genetic disorders, diagnosing infections

39
Q

Requirements to Perform PCR

A

thermocycler; reagents- template DNA to be copied, two single stranded DNA primers, Taq polymerase, deoxynucleotide triphosphates (dNTPs); a small tube with reagents mixed in the proper concentrations placed in the thermocycler

40
Q

Thermocycler is programmed to cycle through a series of temperature changes…

A

melting step- high temp (95-99C) to separate double stranded DNA; annealing step- lower temp (50-65C) allows the primers to anneal with the template DNA; Extension step- optimal temp (65-75C) for the DNA polymersase to copy the target DNA

41
Q

Real Time PCR

A

modified PCR that uses fluorescence imaging to visualize DNA copies as they are made; computer programs allow technicians to see the data immediately or in real time; sometimes called quantitative PCR (qPCR) (can measure how many copies of the target gene were initially present in the sample)

42
Q

Reverse Transcription PCR

A

RT-PCR; useful for detecting RNA in a sample; requires reverse transcriptase (builds DNA that is complementary to target RNA molecules in a sample); ex) Covid 19 tests detects the genome of the virus

43
Q

Genetic Information Nondiscrimination Act of 2008

A

GINA; attempt to protect pateints from discrimination based on genetic information; act is fairly weak; provides a number of loopholes for insureres and employers

44
Q

Recombinant DNA Techniques

A

provide a way to insert a desired gene into an expression system; allows particular proteins to be produced in large amounts; DNA is generated or engineered by combining DNA from different organisms

45
Q

Recombinant DNA Step 1:

A

Gene isolation and copying; building a DNA construct starts with isloating the gene that encodes the desired protein; PCR primers can be designed that flank the desired gene; PCR copies the desired gene through a series of thermocycles

46
Q

Recombinant DNA Step 2:

A

inserting the desired gene into a plasmid; once the desired gene is copied, it is inserted into a cloning vector (cloning vectors are commercially available); desired gene copies and plasmid are cut with a restriction enzyme to generate sticky ends; resulting compatible sticky ends are joinged using DNA ligase; forms a completed recombinant vector

47
Q

Recombinant DNA Step 3:

A

transforming the plasmid into cells for expression; newly made rDNA construct is inserted into host cells; prokaryotic cell lines are often used because they are usually cheap to culture and maintain; eukaryotic cells may be needed inc ertain cases due to certain protein modifications; host cells produce large quanities of the desired protein

48
Q

CRISPR-Cas 9

A

gene editing tool; can locate a specific DNA sequence and cut it out with surgical precision; new DNA can be plugged into the cut site

49
Q

CRISPR-Cas 9 Components

A

CRISPR- serves as a GPS for finding the desired genetic sequence that is to be cut out; Cas9 Enzyme- scalpel that cuts the DNA sequence once it is located

50
Q

How CRISPR works…

A

guide RNA pairs with complementary target DNA; pairing signals the Cas9 enzyme to cut both strands of the DNA; generates a double strand break; new DNA can be added by delivering a desired sequence flanked by sequences in the area that have the double strand break; desired sequence is inserted

51
Q

Gene Therapy

A

viruses used as gene delivery agents; adenoviruses and retrovirses are the most commonly used virsues; replacing mutated or missing genes with normal genes could help to control or even cure a disease

52
Q

How Gene Therapy Works…

A

viruses are genetically engineered to be non pathogenic, but remain infectious; desired gene is then packaged into the virus; gene carrying virus is then introduced into the patient; virus enters a human cell; delivers the normal gene to host cell nucleus; normal gene is transcribed and translated

53
Q

Pathogenicity Islands

A

regions of the pathogen genome that encode toxins, virulence factors and resistance mechanisms

54
Q

Gene Microarrays

A

utilizes tools for investigating differences between healthy and diseased cells; utilizes complementary base pairing between nucleotides

55
Q
A

Microbiology (9 decks)

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