Transcript Slide 1 - Cal State LA - Instructional Web Server

Aim:
To develop a new one-step RT-PCR assay to detect H1N1
by designing new primer to target NP gene
Experimental approach:
-nasopharyngeal swabs from humans
-influenza antigen detection kit
-Viral RNA extraction
-RT-PCR
-Gel electrophoresis and UV fluorescence
Background:
-Need adequate methods for diagnosis of emerging
viruses, H1N1
-General molecular diagnosis two-phase process
-Screening of conserved genes: M gene, NP gene
-hemi-nested PCR: targets NP gene, however,
mismatches with primers and published sequences and
long turnaround times
-Seeplex RT-PCR kit: targets M gene, however, at time
of outbreak manufacturer had not disclosed M primer
sequence
-CDC kit: targets M gene, however, did not become
available until later
New Kit
Targeting NP
Main Findings:
-New NP primer design results
in a more sensitive assay
-New kit less sensitive to
seasonal influenza
-New kit comparable sensitivity
at detecting H1N1 as CDC kit
Figure:
-RT-PCR
Shows increased sensitivity in
new assay vs. hemi-nested
assay based on new NP primer
-Graph
Shows comparable sensitivity
of new assay to CDC kit
Hemi-nested Kit
Targeting NP
CDC kit
Critique:
-labeling of figure: weight ladder and
unexplained product
-RNA control for viral RNA extraction
-RNA control for RT-PCR reaction
Discussion:
-unexplained product in gel could be primer dimers
-CDC kit is used as a reference to compare new kit
with real time RT-PCR
-Seeplex kit M primer sequence was not released
during outbreak because manufacturer wanted people
to buy their kit
RT-PCR: RT
mRNA
RT
RT
RT
1. Random primers bind
to mRNA
Reverse Transcriptase
synthesizes first strand
cDNA
mRNA
cDNA
2. Single strand
cDNA/mRNA hybrid
mRNA
cDNA
3. Reverse Transcriptase
can digest mRNA &
synthesize second
strand cDNA
cDNA
cDNA
4. Double strand
cDNA
RT-PCR: PCR
72*C
62*C
95*C
25*C
Taq
Taq
Taq
Taq
Taq
Taq
Taq
Taq
Repeat
DNA
1.
3.
2.
Anneal
Denature
Extend
Template
Steps
complement
primers
template
1-3
PCR Products
strand
30
Cycles
PCR products
500
400
300
200
100
References:
Kevin Chau, Advanced Cell Biology Presentation RT-PCR and gel
electrophoresis animation, March 2010.