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N.Naranbold, National Influenza Laboratory, National Center of Communicable Diseases, Mongolia

COMPARISON OF r-RT-PCR AND MDCK CELL CULTURE FOR DETECTION OF INFLUENZA VIRUSES IN CLINICAL SAMPLES. N.Naranbold, National Influenza Laboratory, National Center of Communicable Diseases, Mongolia. The 10th International Congress of the Asian Society of

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N.Naranbold, National Influenza Laboratory, National Center of Communicable Diseases, Mongolia

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  1. COMPARISON OF r-RT-PCR AND MDCK CELL CULTURE FOR DETECTION OF INFLUENZA VIRUSES IN CLINICAL SAMPLES N.Naranbold, National Influenza Laboratory, National Center of Communicable Diseases, Mongolia The 10th International Congress of the Asian Society of Clinical Pathology and Laboratory Medicine September 10-11, 2009 in Ulaanbaatar Mongolia.

  2. Introduction Rapid detection and identification of causal agents is essential for timely actions to control of outbreaks and successful clinical management of the disease cases.

  3. Introduction However the traditional methods of virology are time-consuming and costly, making them less successful for practicising epidemiologists and clinicians, especially to control short-duration infections like influenza.

  4. Background In this report we share with you our results in the last 2 years with real-time Reverse-transcriptase (r-RT) PCR for this purpose.

  5. Background In our laboratory, we have been introduced real time PCR since 2008. The study aim is to compare r-RT PCR and traditional method to satisfy epidemiological and clinical needs. We are trying to find out sensitive, rapid and cheaper methods for the reliable detection of A (H3, H1) and B influenza viruses.

  6. Methods and Materials • Samples • 565 clinical nasal samplesfrom patients with ILI collected in ISSSs from BaganuurDistrict, UB and Selengheaimag in 2008/2009 cold season. • r-RT-PCR • Instrument (ABI 7300) • RNA purification (QUIAGEN mini RNA kit) • Primer (Invitrogen®A (H1), A(H3), B, M? • Probe?! (TaqMan) • Master-mix (Invitrogen) • Traditional method • Influenza virus isolation (MDCK cell culture) • Serological tests (HA and HIT)

  7. Traditional influenza detection method Specimen collection Virus Isolation on MDCK cell culture Detection: HA Subtyping: HIT ~6-7days

  8. MDCK cell culture ready for virus inoculation

  9. MDCK cell culture with virus CPE

  10. r-RT-PCR influenza detection method Specimen collection RNA isolation r-RT-PCR 4-5 hours

  11. r-RT-PCR Thermal cycler protocol

  12. r-RT-PCR Results

  13. Detection percentage

  14. Duration

  15. Expenses

  16. Conclusion > r-RT-PCR Traditional method (virus isolation on cell culture) • 6 times more sensitive • 30 times quicker • 2.5 times cheaper for influenza virus detection

  17. Conclusion On the basis of the obtained results r-RT_PCR is recommended for routine influenza virus surveillance.

  18. Thank you!

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