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KMID : 0613820140240040361
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Journal of Life Science
2014 Volume.24 No. 4 p.361 ~ p.369
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Development of Broad-range and Specific 16S rRNA PCR for Use in Routine Diagnostic Clinical Microbiology
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Kim Hyun-Chul
Kim Yun-Tae
Kim Hyo-Gyeong
Lee Sang-Hoo
Lee Kyoung-Ryul
Kim Young-Jin
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Abstract
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Broad-range and specific 16S rRNA gene PCR is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. We describe the development of a broad-range and specific PCR primer, based on bacterial 16S rRNA, for use in routine diagnostic clinical microbiology services. The primers were designed by using conservative regions of 16S rRNA sequences from 10 strains. Ninety-eight clinical strains were isolated from clinical patient specimens. A total of 98 strains of bacteria were identified by phenotypic methods; PCR with newly
designed primers and universal primers. All purified PCR products were sequenced using both forward and reverse primers on an automated DNA analyzer. In this study, we evaluated the usefulness of the newly designed primers and the universal primers for the detection of bacteria, and both these techniques were compared with phenotypic methods for bacteria detection. When we also tested 98 strains of clinical isolates with newly designed primers, about 778 bp DNA fragments were amplified and identified from all strains. Of the 98 strains, 94 strains (95.9%) correspond in comparison with
phenotypic methods. The newly designed primers showed that the identities of 98 (100%) strains were the same as those obtained by universal PCR primers. The overall agreement between the newly designed primers and universal primers was 100%. The primer set was designed for rapid, accurate, and cheap identification of bacterial pathogens. We think the newly designed primer set is useful for the identification of pathogenic bacteria.
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KEYWORD
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16S rRNA, clinical isolates, the newly designed primers, universal primers
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