M5 Flashcards
the gold standard for the detection of SARS-CoV-2 due to the sensitivity and specificity
of the test.
Reverse-transcription PCR (RT-PCR)
Two most common tests used to confirm COVVID19:
RTPCR and ELISA
powerful diagnostic tool in medicine. This technology rapidly and
reliably amplifies specific sections of DNA
PCR
____, allows a diagnostic data that converts RNA to DNA
and then amplify copies of the viral genome that are present.
A modified PCR known as RT-PCR
Examples of RNA Viruses
- Dengue Virus 4. Ebola Virus Disease 7. Measles
- Hepatitis C and E 5. Rabies 8. COVID-19
- West Nile Fever 6. Polio
Each PCR cycle (D → A → E) ____ the amount of the target DNA in less than ___
doubles, five
minutes
___ cycles may be required to produce enough DNA for analysis
20 to 40
primers are combined into a single reaction on the PCR
Multiplex reaction
Multiplex tests also allow an internal
control to be amplified in every ___single reaction.
single
The control is often a common DNA
sequence found in humans, called a ____, that indicates whether or not
the experiment was successful
“housekeeping gene”
PCR was invented by ___ in ___
Dr. Kary Mullis in 1984.
He recognized that he could replicate DNA in vitro using ____ and ___ in a process similar to DNA replication
in a cell’s nucleus
short, synthetic DNA
oligonucleotides (primers) and DNA Polymerase I
____ father of DNA studies; assisted by Rosalind Franklin
Watson and Crick
This technology was then supercharged by ___
Dr. Randy Saiki
He began using thermostable DNA polymerase from the bacteria ____ in PCR reactions
Thermus aquaticus called
Taq
Use of Taq polymerase in PCR was announced by ___
Henry Erlich
The announcement as made in a meeting in___ on ___, submitted for
publication in October 11987, and was published early the next year
Berlin on September 20, 1986
The patent for PCR
with Taq polymerase was filed on ___, and was issued on October 23, 1990
June 17, 1987
Purified double-stranded DNA is mixed with primers, Taq
polymerase, and nucleotides
DENATURATION:
94 c
DENATURATION:
Cool sample to 45-60⁰C to allow primers to base pair with the target
DNA sequence
ANNEALING:
Temperature is raised to 72⁰C, the optimal temperature at which Taq
polymerase will extend the primer to synthesize a new strand of DNA for analysis
EXTENSION
ability to test to correctly identify an
individual with a disease as positive
sensitivity
Ability of a test to correctly identify an
individual without the disease as negative
specificity
Technique used to analyze amplified DNA
Agarose Gel Electrophoresis
The RNA is reverse transcribed into
complementary DNA (cDNA), using __
reverse transcriptase.
SARSCOV2 has ___ main structural proteins.
four
The single stranded
DNA molecule is then completed by the ______ activity of the
reverse transcriptase into cDNA
DNA-dependent DNA polymerase
Monomers of the nucleocapsid of N protein link together to form a ___ which wraps
around and protects the RNA genome.
helical capsid
Embedded in the membrane are several viral proteins: ____
the spike or S protein, the envelope
or E protein, and the membrane or M protein.
The____protein coordinates interactions between the other viral proteins and the
host cell factors, turning cells into virus factories
membrane
As a ___, the envelope protein binds to itself to form channels that facilitates viral
release.
viroporin
The
____ protein binds with human cell surface protein allowing the virus to inject its genetic
material into its host cell
spike
. An intermediate or inconclusive test occurs
when only one of the two SARSCOV targets amplify.
presumptive positive
All buffers except ___ and ____ in this kit can be stored at 15⁰C - 35⁰C
Proteinase K and Carrier molecule
Proteinase K should be stored at ____
2⁰C – 4⁰C after reception.
Carrier molecule should be stored at P_____. Carrier molecule is
not recommended to be frozen and thawed more than ___
-25⁰C to -15⁰C after dissolution, 8 times
in dry sample, Put the dry swab into ___of Swab/Stool lysis buffer and vortex thoroughly.
1 mL
Prepare 0.4% DTT solution.
in sputum
Put ____of fecal sample into 1.5 ml tube
180-220 mg or 200 uL
10 minutes incubation
dry sample
fecal sample treatment
In fecal sample, Centrifuge for ____at ___
3 minutes at 13000 rpm 5
pre treated sample
200 ul
microcentrifuge
1.5 ml
ksb buffer
250 ul
proteinase k
200 ul
carrier molecule
5 ul
Vortex thoroughly for ___ to mix and Incubate at___ for
10 seconds, 56⁰C for 10 min
Absolute ethanol Vortex
350 uL
After dispensing all the mixture on the provided spin column, centrifuge at ___ for __
10,000 rpm for
1 minute.
● Unicellular
● Can survive without host
● Have DNA
bacteria
Unicellular
● Cannot survive without host
● Only classified as RNA or DNA
● Many viruses don’t have DNA
virus
Was awarded Nobel Prize Chemistry in 1993
- Used fragments of E.coli DNA polymerase to
describe the in-vitro amplification of genes
KARY MULLIS, Ph. D
First proposed by___
H.G. Khorona, et al (1970s)
used in diagnostic PCR
MULTIPLE PRIMERS
Base lane
● Intended for analyte of interest
lane 1
SARS-COV 1
- 1992-2002
MERS-COV
- 2012 peak
