Evaluation study of real-time PCR for the rapid detection of respiratory virus in pediatric patients

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Evaluation study of real-time PCR for the rapid detection of respiratory virus in pediatric patients

 

Author: Lee, Hon-wang
Title: Evaluation study of real-time PCR for the rapid detection of respiratory virus in pediatric patients
Degree: M.Sc.
Year: 2008
Subject: Hong Kong Polytechnic University -- Dissertations.
Respiratory infections.
Infection in children -- Diagnosis.
Polymerase chain reaction.
Department: Dept. of Health Technology and Informatics
Pages: iv, 71 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2305605
URI: http://theses.lib.polyu.edu.hk/handle/200/5100
Abstract: Lower respiratory tract infection (LRTI) is still one of the most common diseases found in pediatric patients. The most common agents causing LRTI include Influenza A virus and respiratory syncytical virus(RSV). These viruses cause morbidity and mortality annually among children and the elderly. The aim of this study was to establish a real-time PCR assay for the rapid detection of multiple infective agents causing LRTI from nasopharyngeal aspirates (NPA). Primer sets for Influenza A, RSV, rhinovirus and human metapneumovirus (HMPV) were selected to perform real-time PCR assay to screen for the respective pathogens in pediatric patients. Corresponding primers sets were chosen for the most conservative regions of interest. For HMPV and RSV, the region of nucleoprotein (n gene) was chosen. The 5' non-coding regions (NCR) were selected for rhinovirus, and segment 7 matrix gene was also selected for Influenza A virus. All these region have been shown to be conserved among the gene sequence of interest. A total of 120 NPA samples come from children with acute respiratory diseases were retrospectively tested. The result obtained from real-time PCR was compared with the result from viral culture which was conducted by Public Health Laboratory Centre. The qualitative results of the real-time PCR were in good agreement with conventional viral culture. Of the 28 cases of Influenza A, 10 cases of RSV and 2 cases of rhinoviruses detected using tissue culture techniques. Real-time PCR detection recognized all Influenza A, RSV and rhinovirus with nearly 100% congruity. Furthermore, the method of real-time PCR detected the presence of Influenza A virus in 5 additional specimens, and in one additional case of RSV and 6 additional cases of rhinovirus. The overall positive rates in this study were 27.5%, 9.2% and 6.7% for Influenza A, RSV and rhinovirus respectively. When compared with the positive rate of viral culture, 23.3%, 8.3% and 1.7% for Influenza A, RSV and rhinovirus respectively, the result was superior. The data obtained was also comparable to figures obtained in previous studies. Performing real-time PCR is preferable to conventional PCR and viral culture as the assay produces fluorescence-labeled PCR products, which can be measured and recorded during PCR cycling, thus reduce time-consuming post-PCR process, such as gel electrophoresis, blotting, and hybridization. Elimination of post-PCR processing increases the speed of the assay and reduces the chances of cross-contamination. This evaluation study proved that the method of real-time PCR is more sensitive than conventional tissue culture isolation, with corresponding specificity. This technique is valuable for surveillance and rapid identification of Influenza A, RSV, and rhinovirus for early detection and appropriate management of pediatric cases. Early recognition of viral etiological agents can also help prevent inappropriate antibiotic therapy, thus reducing selective pressure for resistance development.

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