| Co-Investigator(Kenkyū-buntansha) |
YAMASHITA Yoshihisa Kyushu University, Faculty of Dental Science, Professor, 大学院・歯学研究院, 教授 (20192403)
MAENO Masao Nihon University, School of Dentistry, Professor, 歯学部, 教授 (60147618)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from "pneumococcuslike" oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search fur differences between Streptococcus pneumoniae strain WU2 and the most closely related oral streptococcus, Streptococcus mitis strain 903. We identified 19 clones that contained S.pneumoniae-specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of S.mitis and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of S.pneumoniae were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four S.pneumoniae clinical isolates and from 20 atypical organisms classified as S.mitis or S.oralis, which harbored genes encoding the pneumococcal virulence factors autolysin (lytA) or pneumolysin (ply), a
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s templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of S.pneumoniae infections (J Clin Microbiol 2005;43;4528-4534).
A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63℃) with high specificity, efficiency and rapidity, was examined regarding its applicability for detecting S.pneumoniae. An S.pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S.pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S.oralis, 17 S.mitis and 1 Streptococcus species) that harbor virulencefactor-encoding genes (lytA or ply) were tried to differentiate S.pneumoniae. The detection of S.pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S.pneumoniae infection (J Clin Microbiol 2005;43:1581-1586).
It is difficult and time-consuming to distinguish Haemophilus influenzae (Hi) from the genotypically similar H.parainfluenzae, and serotyping of the capsular antigen of Hi is troublesome owing to ambiguous results. LAMP was evaluated for detecting Hi and Haemophilus influenzae type b (Hib). Two LAMP primer sets targeting the outer membrane protein p6 gene and Hib-specific capsulation locus were designed. The specificity of each primer set was validated. Within 60 min, LAMP detected purified DNA with over 10-fold greater sensitivity than did conventional PCR. Therefore, LAMP may represent a rapid, sensitive, and reliable means of diagnosing Hi and Hib infection (FEMS Microbiol lett 2005 ; submitted). Less
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