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A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene  Stig Ove Hjelmevoll,

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Presentation on theme: "A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene  Stig Ove Hjelmevoll,"— Presentation transcript:

1 A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene  Stig Ove Hjelmevoll, Merethe Elise Olsen, Johanna U. Ericson Sollid, Håkon Haaheim, Magnus Unemo, Vegard Skogen  The Journal of Molecular Diagnostics  Volume 8, Issue 5, Pages (November 2006) DOI: /jmoldx Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Nucleotide sequence alignment of partial N. gonorrhoeae consensus porA pseudogene compiled from 87 sequences34 and the corresponding N. meningitidis sequence. The boxed sequences on the flanks are the forward and reverse primer location, respectively. The boxed sequence in the middle is the location of the probe. The MC58 sequence is the partial porA sequence of the previously published whole-genome sequenced N. meningitidis strain MC58.42 The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Dilution series of commercially purified N. gonorrhoeae DNA (Tebu-bio) and detection in the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study. The six 10-fold dilutions showed good linearity. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 Standard curve based on the dilution series in Figure 2. The slope is the basis for the amplification efficacy (E) calculation. In a 100% efficient PCR, the DNA will double per cycle or use cycles to increase DNA copies by 10-fold (23.33 ≈ 10). The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

5 Figure 4 IAC amplification with low-level N. gonorrhoeae. The figure shows the internal amplification control amplified in dilutions of increasing concentrations of N. gonorrhoeae DNA. The amplification lines that do not cross the thick line are negative due to larger amounts of N. gonorrhoeae DNA. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

6 Figure 5 Dilution series of N. gonorrhoeae DNA using two parallels from master mixes with and without IAC. There was no significant difference in the amplification of N. gonorrhoeae DNA between the two master mixes as all four parallels are approximately equal. The mastermix without IAC shows a slightly higher ΔRn than the master mix with IAC. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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