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Download Slide 1 - Cal State LA - Instructional Web Server
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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.
