A one-step real-time reverse transcription PCR (RT-PCR) assay was developed for the detection of human rhinovirus (RV) in nasal aspirate (NA) specimens. A set of primers was designed to amplify a 120-base target in the 5' non-coding region of RV RNA. The amplicon was detected using TaqMan minor groove binder (MGB) probes and ABI Prism sequence detectors.
Three probes were evaluated using stock suspensions of 15 RV strains representing serotypes 1A, 2, 3, 7, 17, 21, 29, 37, 39, 40, 58, 62, 66, 72, and 87. The initial probe that was designed, Pic-4, detected 11 of the RV strains. Probe Pic-6 was designed with a degeneracy at the first 5' nucleotide to accommodate a target-probe mismatch. Although Pic-6 improved the detection limit for some strains, detection of RVs containing multiple mismatches was unsatisfactory. Probe Pic-5 was designed with nucleotide degeneracies at three positions to account for multiple mismatches. Pic-5 detected 14 of the RVs at a lower limit of detection of 0.00001 to 0.01 50% tissue culture infectious dose (TCID50) equivalents/PCR reaction. RV-87, recently classified as an enterovirus (EV), was not detected with any of the probes.
The assay with Pic-5 detected 30 plus or minus 50 copies of a plasmid containing an RV-2 target sequence. The assay yielded negative results when nucleic acids from human cells, EVs, and a panel of bacteria and viruses found in the human nasopharynx were tested. The assay with Pic-4 yielded a detection limit of 1.0 TCID50 equivalents/PCR reaction in both neat and supernatant NA specimens seeded with RV-2.
A total of 48 NA samples obtained from children with cold-like symptoms were tested by the RT-PCR assay with probe Pic-5; 8 (16.7%) of the samples were positive.
In conclusion, the real-time TaqMan MGB RT-PCR assay with Pic-5 is rapid, sensitive, and specific and has the capability of detecting at least 14 serotypes of RV. The public health significance of this study is that the RT-PCR assay with Pic-5 may lead to improvements in the diagnosis and surveillance of RV infections, and to a better understanding of the ability of RV to cause serious infection in immunocompromised patients.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07252005-130338 |
| Date | 13 September 2005 |
| Creators | Do, Duc Hoang |
| Contributors | Robert M. Wadowsky, Sc.D., Lee H. Harrison, M.D., Frank J. Jenkins, Ph.D. |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-07252005-130338/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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