OBJECTIVETo establish the specific 16S-23S rRNA gene spacer regions in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis.

METHODSA pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA from sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 26 species was amplified by PCR, and further analyzed by RFLP, DNA cloning and sequences analysis. Furthermore, all specimens were examined by bacterial culturing and PCR-RFLP analysis. The evaluation of these assays in practical clinic practice was also discussed.

RESULTSRestriction enzyme analysis revealed one, two or three bands or more observed among the 26 different standard strains. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction with human genomic DNA, fungus or virus. Fourteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. The only difference between K.pneumoniae and E. durans was located at the site of the 779th nucleotide according to the sequence analysis and only XmaIII digestion could distinguish one from another. Of 42 specimens from septicemic neonates, 15 were identified as positive by blood culture at a rate of 35.7%. However, 27 specimens identified as positive by PCR, with a rate of 64.2%, a method significantly more effective than blood culture (P < 0.01). Of 6 cerebrospinal fluid (CSF) specimens, one tested positive for S.epidermidis was also positive by PCR, two culture negative were positive by PCR and diagnosed as S.epidermidis according to the DNA pattern. One positive for C.neoformans was negative by PCR. The other two specimens were negative by both PCR and culture.

CONCLUSIONSThe method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in providing a new technique for detecting pathogens in clinical bacterial infections.

Bacteria ; genetics ; isolation & purification ; DNA, Bacterial ; analysis ; chemistry ; DNA, Ribosomal ; analysis ; chemistry ; Genes, rRNA ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Sensitivity and Specificity ; Sequence Analysis, DNA

OBJECTIVETo establish the specific 16S-23S rRNA gene spacer regions pattern in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis.

METHODSA pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA of sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 27 species was amplified by PCR, and further studied by RFLP, DNA cloning and sequences analysis. Meanwhile, all specimens were examined by bacterial culturing and PCR-RFLP analysis.

RESULTSThe 27 different standard strains showed one, two, three or more than three bands. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction to the human, fungal or viral genomic DNAs. Fifteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. Klebsiella pneumoniae (Kp) and Enterococcus durans (Ed) could not be differentiated from each other by Alu I or Hinf I digestion. The spacer sequences of the Kp and Ed were 908 bp and 909 bp, respectively, and they differed only at the site of the 779th nucleotide. The former was G, and the latter was A. The 760 - 790 bp sequence of Kp was as follows: CGACTGCACCGCCTCCTAC / GGCCGCGTATTC. The 760 - 790 bp sequence of Ed was as follows: CGACTGCAC CGCCTCCTAC / AGCCGCGTATTC. Only one enzyme XmaIII, could discriminate the two. The cleaving site of XmaIII is C downward arrow GGCCG. Kp DNA was cleaved into 778 bp and 130 bp fragments, while E. durans was not. Of 42 specimens with suspected septicemia, 15 were positive (35.7%) on blood culture, and 27 on PCR (64.29%). The positive rate of PCR was significantly higher than that of blood culture (P < 0.01). Of the six CSF specimens, one was positive for Staphylococcus epidermidis (Se) on culture as well as by PCR, while two specimens which were negative on cultures were positive by PCR and were diagnosed as Se according to its DNA pattern. One specimen was culture-positive for Cryptococcus neoformans (Cn) but was negative by PCR. The other two specimens were negative by both PCR and culture. Fifteen blood samples from healthy children were negative by both blood culture and PCR.

CONCLUSIONSThe method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in detecting pathogens in clinical bacterial infections.

Bacterial Infections ; diagnosis ; microbiology ; DNA, Bacterial ; chemistry ; genetics ; DNA, Ribosomal Spacer ; genetics ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Sequence Analysis, DNA

OBJECTIVETo set up a rapid and simple method for identificating bacteria by 16S-23SrDNA intergenic spacer regions (ISRs).

METHODSPolymorphic products of PCR from ISRs were selected on agarose gel and sequenced directly using purified fragments by excising the gel without cloning. Nucleotide sequences were compared with GenBank databases and analyzed by DNAMAN program.

RESULTSThere was only a single product in streptococcus genus after PCR amplification of 16S-23SrDNA ISRs. Five streptococcal species were obtained from 7 strains of streptococcus. Two major amplicons were consistently generated for 8 strains of Haemophilus influenzae (H. influenzae). The sequence data showed that they all belonged to H. influenzae type b on GenBank databases.

CONCLUSIONPCR and direct sequencing of 16S-23SrDNA ISRs were very successful methods for bacterial species identification.

Base Sequence ; DNA, Ribosomal Spacer ; chemistry ; Haemophilus influenzae ; genetics ; isolation & purification ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Streptococcus ; genetics ; isolation & purification

The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.
Bacteremia ; diagnosis ; genetics ; Bacterial Typing Techniques ; Bacteriological Techniques ; DNA Probes ; Genetic Techniques ; Listeria monocytogenes ; metabolism ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis ; Oligonucleotides ; chemistry ; RNA, Ribosomal ; chemistry ; RNA, Ribosomal, 23S ; genetics ; Stem Cells

OBJECTIVETo establish polymerase chain reaction (PCR) technique for the detection of specific genes related to species of genus Bartonella, and for diagnosing clinically suspected cat-scratch disease (CSD) case complicated with pneumonia on both lungs. The appearance of Bartonella infectious diseases calls for genus and species detection and tools for identification in order to make clinical diagnosis and carry on epidemiological studies.

METHODSOne pair of primer TIle.455p-TAla.885n was designed based on the fact that tRNA(Ile)-tRNA(Ala) intergenic spacer region in 16S-23S rRNA intergenic spacer (ITS) of genus Bartonella were high variable sequences flanked by completely conserved tRNA-encoding genes. 16S-23S rRNA was longer than that which had been described in other bacteria. Two published pairs of primers were used to directly detect the specific gene fragments of Bartonella species DNA extracts from human blood, followed by PCR product Sequencing and nucleotide base sequence analysis.

RESULTSAmplification products of the three pairs of primers had the same predicted size of those in Bartonella spp. According to the different length of electrophoresis bank, the sample was identified as a species of genus Bartonella other than the positive control. Sequence analysis showed that the nuleotide sequence from the PCR product of primer TIle.455p-TAla.885n was identical to the Bartonella isolated from Yunnan in China.

CONCLUSIONSPCR-based assay provided a simple and rapid means to detect pathogenic Bartonella species in humans and mammalian hosts as well as in arthropod vecters. This study suggested that this pathogenic Bartonella species existed in patients in northern and southern parts of China.

Animals ; Bartonella ; genetics ; isolation & purification ; Bartonella Infections ; diagnosis ; microbiology ; Base Sequence ; Cat-Scratch Disease ; diagnosis ; microbiology ; Cats ; China ; Diagnosis, Differential ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; RNA, Transfer, Ala ; chemistry ; genetics ; RNA, Transfer, Ile ; chemistry ; genetics ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid

Using three reference strains of Brachyspira hyodysenteriae (B204, B234, B169), one B. pilosicoli (P43/6/78), one B. murdochii (56-150), one B. intermedia (PWS/A), one B. innocens (B256) and ten Korean isolates, PCR-RFLP analysis of DNA encoding 23S rRNA was performed to establish a rapid and accurate method for characterizing porcine intestinal spirochetes. Consequently, B. hyodysenteriae and B. pilosicoli revealed different restriction patterns; however, the other three species shared the same pattern. These findings are not consistent with a prior report. Differences in 23S rRNA gene sequences, between two B. murdochii strains, 56-150 and 155-20, were observed. These results indicate that 23S rRNA PCR-RFLP could be used as an identification method for pathogenic Brachyspira spp. (B. hyodysenteriae and B. pilosicoli) as well as an epidemiological tool for characterizing spirochetes isolated from swine.
Animals ; DNA, Bacterial/genetics ; Dysentery, Bacillary/diagnosis/microbiology/*veterinary ; Korea ; Phylogeny ; Polymerase Chain Reaction/methods/*veterinary ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 23S/chemistry/*genetics ; Spirochaetales/*genetics/*isolation&purification ; Spirochaetales Infections/diagnosis/microbiology/*veterinary ; Swine ; Swine Diseases/diagnosis/*microbiology

OBJECTIVETo define the main genotypes in Guizhou agricultural areas by molecular epidemiologic investigation of 21 Borrelia burgdorferi sensu lato of Lyme disease spirochetes and to provide the scientific bases for formulating a preventive policy.

METHODSPolymerase chain reaction (PCR) technique was used to amplify the 23S(rrl)-5S(rrf) intergenic spacer, and amplified products were analyzed by restriction fragment length polymorphism (RFLP) and nucleotide sequencing.

RESULTSThere were two genospecies in the strains: 20 strains belong to Borrelia valaisiana, 1 strain is Borelia sp.

CONCLUSIONBorrelia valaisiana was the main genotype in Guizhou agricultural areas. The harmness of B. valaisiana to human being has been confirmed. In order to efficiently prevent the harmness of agent to the people in Guizhou agriculture areas, we should study the risk further.

Base Sequence ; Borrelia burgdorferi ; classification ; genetics ; China ; DNA, Bacterial ; chemistry ; genetics ; DNA, Ribosomal Spacer ; genetics ; Humans ; Lyme Disease ; microbiology ; Molecular Sequence Data ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 23S ; genetics ; RNA, Ribosomal, 5S ; genetics ; Ribotyping ; Sequence Analysis, DNA

A slowly growing, non-chromogenic mycobacterial strain was isolated from sputum and bronchial lavage fluid samples of a patient presenting with productive cough, blood-tinged sputum, low-grade fever, and weakness. A positive acid-fast bacilli sputum smear result prompted the initiation of an anti-tuberculosis regimen. Multiplex real-time PCR showed a negative result for Mycobacterium tuberculosis complex and a positive result for nontuberculous mycobacteria. The DNA chip test confirmed this organism as a member of the genus Mycobacterium, but could not specify the species. Interestingly, the mycolic acid patterns obtained by HPLC nearly overlapped with those of M. simulans. The sequences of the Mycobacterium 16S rRNA gene and 16S-23S internal transcribed spacer region were unique and were found to have 100% similarity with those of M. riyadhense. After a review of the literature, we report this case as the first Korean case of M. riyadhense lung infection.
Adult ; Antitubercular Agents/pharmacology ; Chromatography, High Pressure Liquid ; Female ; Humans ; Lung Diseases/*microbiology ; Microbial Sensitivity Tests ; Mycobacterium/classification/drug effects/*isolation & purification ; Mycobacterium Infections/microbiology ; Mycobacterium tuberculosis/genetics/isolation & purification ; Mycolic Acids/analysis ; Oligonucleotide Array Sequence Analysis ; Phylogeny ; RNA, Ribosomal, 16S/chemistry/genetics ; RNA, Ribosomal, 23S/chemistry/genetics ; Republic of Korea ; Sequence Analysis, DNA