A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control
<p>Abstract</p> <p>Background</p> <p>Avian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential trans...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2006-05-01
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| Series: | BMC Infectious Diseases |
| Online Access: | http://www.biomedcentral.com/1471-2334/6/87 |
| _version_ | 1818492615654375424 |
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| author | Delogu Mauro De Marco Maria Chiappini Barbara Campitelli Laura Donatelli Isabella Bedini Barbara Di Trani Livia Buonavoglia Canio Vaccari Gabriele |
| author_facet | Delogu Mauro De Marco Maria Chiappini Barbara Campitelli Laura Donatelli Isabella Bedini Barbara Di Trani Livia Buonavoglia Canio Vaccari Gabriele |
| author_sort | Delogu Mauro |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Avian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results.</p> <p>Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC.</p> <p>Methods</p> <p>RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of <it>in vitro </it>transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted.</p> <p>Results</p> <p>The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 10<sup>8 </sup>copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR.</p> <p>Conclusion</p> <p>The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens.</p> |
| first_indexed | 2024-12-10T17:45:28Z |
| format | Article |
| id | doaj.art-3cb40015b351424e8e4ab99b3f0b443a |
| institution | Directory Open Access Journal |
| issn | 1471-2334 |
| language | English |
| last_indexed | 2024-12-10T17:45:28Z |
| publishDate | 2006-05-01 |
| publisher | BMC |
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| series | BMC Infectious Diseases |
| spelling | doaj.art-3cb40015b351424e8e4ab99b3f0b443a2022-12-22T01:39:15ZengBMCBMC Infectious Diseases1471-23342006-05-01618710.1186/1471-2334-6-87A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive controlDelogu MauroDe Marco MariaChiappini BarbaraCampitelli LauraDonatelli IsabellaBedini BarbaraDi Trani LiviaBuonavoglia CanioVaccari Gabriele<p>Abstract</p> <p>Background</p> <p>Avian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results.</p> <p>Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC.</p> <p>Methods</p> <p>RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of <it>in vitro </it>transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted.</p> <p>Results</p> <p>The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 10<sup>8 </sup>copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR.</p> <p>Conclusion</p> <p>The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens.</p>http://www.biomedcentral.com/1471-2334/6/87 |
| spellingShingle | Delogu Mauro De Marco Maria Chiappini Barbara Campitelli Laura Donatelli Isabella Bedini Barbara Di Trani Livia Buonavoglia Canio Vaccari Gabriele A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control BMC Infectious Diseases |
| title | A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control |
| title_full | A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control |
| title_fullStr | A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control |
| title_full_unstemmed | A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control |
| title_short | A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control |
| title_sort | sensitive one step real time pcr for detection of avian influenza viruses using a mgb probe and an internal positive control |
| url | http://www.biomedcentral.com/1471-2334/6/87 |
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