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Broad-Range Ribosomal RNA Real-Time PCR after Removal of DNA from Reagents: Melting Profiles for Clinically Important Bacteria

¹ School of Medical Technology and Graduate Institute of Medical Biotechnology, Chang Gung University, Tao-Yuan 333, Taiwan, Republic of China;
² Laboratory of Molecular Diagnosis and
⁵ Department of Emergency Medicine, Chang Gung Memorial Hospital, Tao-Yuan 333, Taiwan, Republic of China;
³ Department of Medical Technology, China Medical College, Taichung 404, Taiwan, Republic of China;
⁴ Divisions of Basic Sciences and of Restorative and Prosthodontic Sciences, College of Dentistry, New York University, New York, NY 10010-4086;

^aaddress correspondence to this author at: Department of Emergency Medicine, Chang Gung Memorial Hospital, Kweishan Hsiang, Taoyuan, Taiwan, ROC; fax 886-03-3289355, e-mail sschang@adm.cgmh. org.tw

The first 300 words of the full text of this article appear below.

With the ability to exponentially amplify regions of DNA, two different PCR-based strategies have been developed for nonculture diagnosis of bacteremia. The first approach targets the species-specific genes for amplification (1), whereas the second approach involves universal PCR amplification of conserved bacterial DNA sequences, such as the 16S rRNA, the 23S rRNA, and the 16S-23S rRNA interspace regions (2)(3)(4). Although universal PCR is not able to distinguish bacteria to the species level, numerous studies have shown that this method provides valuable information complementary to the results of time-consuming and subjective phenotypic tests used in detection of bacterial infection (5) and can be used to differentiate bacterial from viral or other infections (6)(7). In clinical applications, real-time PCR for broad-range amplification of bacterial DNA sequences could offer additional benefits: it is less labor-intensive, less time-consuming, and reduces the risk of PCR carryover contamination.

A major obstacle for broad-range PCR amplification is the presence of bacterial DNA in the Taq DNA polymerase and real-time PCR master mixture (8)(9)(10). The contaminating DNA is effectively amplified, giving rise to false-positive results. Efforts have been taken to eliminate contaminating DNA, including the use of Sau3AI restriction endonuclease (11), DNase I (12)(13), and ultraviolet irradiation (7)(8). Although these strategies may be effective for conventional PCR, the study by Corless et al. (14) indicated that the contamination issue could not be avoided without affecting sensitivity when TaqMan probe-based real-time PCR is performed. Therefore, an appropriate method for removal of contaminating bacterial DNA and subsequent real-time amplification is required. In this study, we address this issue and report optimized conditions for broad-range amplification of the . . . [Full Text of this Article]

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				J.-C. Cheng, C.-L. Huang, C.-C. Lin, C.-C. Chen, Y.-C. Chang, S.-S. Chang, and C.-P. Tseng Rapid Detection and Identification of Clinically Important Bacteria by High-Resolution Melting Analysis after Broad-Range Ribosomal RNA Real-Time PCR Clin. Chem., November 1, 2006; 52(11): 1997 - 2004. [Abstract] [Full Text] [PDF]

				H. P. Horz, M. E. Vianna, B. P. F. A. Gomes, and G. Conrads Evaluation of Universal Probes and Primer Sets for Assessing Total Bacterial Load in Clinical Samples: General Implications and Practical Use in Endodontic Antimicrobial Therapy J. Clin. Microbiol., October 1, 2005; 43(10): 5332 - 5337. [Abstract] [Full Text] [PDF]