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This Article Full Text Full Text (PDF) 069286.Supplemental Data All Versions of this Article: clinchem.2006.069286v1 52/11/1997    most recent Submit an electronic Letter to the Editor about this paper Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (8) Citing Articles via Google Scholar Google Scholar Articles by Cheng, J.-C. Articles by Tseng, C.-P. Search for Related Content PubMed PubMed Citation Articles by Cheng, J.-C. Articles by Tseng, C.-P. Related Collections Infectious Disease Molecular Diagnostics and Genetics Automation and Analytical Techniques

Rapid Detection and Identification of Clinically Important Bacteria by High-Resolution Melting Analysis after Broad-Range Ribosomal RNA Real-Time PCR

¹ School of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan, Republic of China.
Graduate Institutes of² Basic Medical Sciences,³ Medical Biotechnology, and ⁴ Clinical Medical Sciences, Chang Gung University, Tao-Yuan, Taiwan, Republic of China.
⁵ Department of Pathology and Laboratory Medicine, Li Shin Hospital, Tao-Yuan, Taiwan, Republic of China.
⁶ Department of Nursing, Chang Gung Institute of Technology, Tao-Yuan, Taiwan, Republic of China.
⁷ Department of Emergency Medicine and ⁸ Laboratory of Molecular Diagnostics, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan, Republic of China

^aAddress correspondence to these authors at: Ching-Ping Tseng, Graduate Institute of Medical Biotechnology, Chang Gung University, 259 Wen-Hwai 1st Rd. Kwei-Shen, Tao-Yuan 333, Taiwan, Republic of China. Fax 886-3-2118355; e-mail ctseng{at}mail.cgu.edu.tw. Shy-Shin Chang, Department of Emergency Medicine, Chang Gung Memorial Hospital, Tao-Yuan 333, Taiwan, Republic of China. Fax 886-3-3287715; e-mail: sschang{at}adm.cgmh.org.tw.

Background: Broad-range PCR provides valuable information for detecting bacterial infections. This study assesses the combined use of broad-range real-time PCR and high-resolution melting analysis for rapid detection and identification of clinically important bacteria.

Methods: We subjected 46 bacterial culture colonies representing 25 clinically important bacterial species to LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye. We performed high-resolution melting analysis of the PCR products with the HR-1 instrument and used melting profiles as molecular fingerprints for bacterial species identification. We validated this method via assessment of 54 consecutive bacteria culture colonies obtained from a clinical microbiology laboratory.

Results: The 16S rRNA gene of all 25 bacterial species was amplifiable by this method, with PCR product lengths of 216 or 217 bp. Of the 25 bacterial species, we identified 11 via a 1-step post-PCR high-resolution melting analysis. The remaining bacterial species were identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species or by a 2nd real-time PCR targeting a different region of the 16S rRNA gene. A high-resolution melting database and a working protocol were established for identifying these 25 bacterial species. In the validation assay, a 94% accuracy rate was achieved when the bacterial species were in the high-resolution melting database.

Conclusions: This assay requires no multiplexing or hybridization probes and provides a new approach for bacterial species identification in a molecular diagnostic laboratory.

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