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Influenza and Other Respiratory Viruses
Volume 4, Issue 5, pages 277?293, September 2010
Original Article: Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs
1. Marek J. Slomka1,
2. Anstice L. E. Densham1,
3. Vivien J. Coward1,
4. Steve Essen1,
5. Sharon M. Brookes1,
6. Richard M. Irvine1,
7. Erica Spackman2,
8. Jonathan Ridgeon1,
9. Rebecca Gardner1,
10. Amanda Hanna1,
11. David L. Suarez2,
12. Ian H. Brown1
Article first published online: 17 AUG 2010
DOI: 10.1111/j.1750-2659.2010.00149.x
Background There is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods.
Objectives First, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections.
Methods RRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v.
Results The ?perfect match? M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a ?gold standard?, while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. ?Perfect match? M gene RRT PCR had 100% sensitivity and 95?2% specificity for swabs, 93?6% and 98?6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99?1%, respectively, for the swabs, and 100% and 100% for the tissues.
Conclusions Two RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated.
Volume 4, Issue 5, pages 277?293, September 2010
Original Article: Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs
1. Marek J. Slomka1,
2. Anstice L. E. Densham1,
3. Vivien J. Coward1,
4. Steve Essen1,
5. Sharon M. Brookes1,
6. Richard M. Irvine1,
7. Erica Spackman2,
8. Jonathan Ridgeon1,
9. Rebecca Gardner1,
10. Amanda Hanna1,
11. David L. Suarez2,
12. Ian H. Brown1
Article first published online: 17 AUG 2010
DOI: 10.1111/j.1750-2659.2010.00149.x
Background There is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods.
Objectives First, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections.
Methods RRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v.
Results The ?perfect match? M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a ?gold standard?, while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. ?Perfect match? M gene RRT PCR had 100% sensitivity and 95?2% specificity for swabs, 93?6% and 98?6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99?1%, respectively, for the swabs, and 100% and 100% for the tissues.
Conclusions Two RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated.
