1. 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

2. 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

3. 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 New Kit Targeting NP Hemi-nested Kit Targeting NP CDC kit

4. 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

5. 4. Double strand cDNA mRNA 1. Random primers bind to mRNA Reverse Transcriptase synthesizes first strand cDNA 3. Reverse Transcriptase can digest mRNA & synthesize second strand cDNA mRNA cDNA mRNA cDNA 2. Single strand cDNA/mRNA hybrid RT RT-PCR: RT

6. DNA Template 25*C95*C62*C72*C 1. Denature template3. Extend complement strand 2. Anneal primersRepeat Steps 1-3 30 Cycles Taq PCR Products RT-PCR: PCR

7. RT-PCR: Gel electrophoresis 100 200 300 400 500 PCR products

8. References: Kevin Chau, Advanced Cell Biology Presentation RT-PCR and gel electrophoresis animation, March 2010.