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Objective:To establish the concatenated DNA sequencing of 16S ribosomal RNA （16S rRNA） and DNA gyrase subunit B （gyrB） gene， and provide evidence for the identification and classification of Vibrio parahaemolyticus （V. parahaemolyticus）. Methods:Typical strains in the genus of Vibrio spp. was selected， such as V. parahaemolyticus， V. alginolyticus and other species for examination of 16S rRNA and gyrB gene as target. Primers were separately designed to amplify these two nucleotide fragments. Phylogenetic analysis was performed using the concatenated sequences. Results:The concatenated 16S rRNA+gyrB nucleotide sequence of V. parahaemolyticus formed a single cluster in the phylogenetic analysis， which identified the typical strains of Vibro spp. at the species level. Conclusion:In our study， an identification method of V. parahaemolyticus is established based on concatenated 16S rRNA+gyrB nucleotide sequencing. It can identify the strains of V. parahaemolyticus at the species level， which may be applied in phylogenetic analysis and contamination tracing of V. parahaemolyticus in food and drug control.
ABSTRACT
BACKGROUND: Among the many Vibrio species that can cause infections in humans, several species can cause a fatal outcome. Therefore, accurate identification of Vibrio species is very important. Since some species show atypical phenotypic features, selecting an appropriate molecular method is necessary to avoid misdiagnosis. METHODS: Vibrio clinical isolates (N=53) and reference strains (N=8) were used in this study. We analyzed the following sequences for identification: dnaJ gene, 16S rDNA, gyrase B (gyrB) V. vulnificus-specific sequence, gyrB V. navarrensis-specific sequence, and V. vulnificus hemolysin gene PCR (Vvh PCR). We performed phylogenetic analysis of the 16S rDNA, dnaJ, and gyrB sequences. Final identification was based on the combined results of all tests described above. Concordance of the 16S rDNA and dnaJ sequence analysis was measured using the Chi-square test. RESULTS: The 61 Vibrio strains were identified as follows, in descending order: V. vulnificus (78.69%), V. parahaemolyticus (6.56%), V. navarrensis (4.92%), V. mimicus (1.64%), V. cholera (1.64%), V. furnissii (1.64%), V. alginolyticus (1.64%), and Grimontia hollisae (1.64%). The accuracy rates of the dnaJ gene and 16S rDNA sequence for identification were 91.80% and 86.89%, respectively. The 16S rDNA and dnaJ sequences showed a concordance rate of 0.45, which indicates moderate agreement. CONCLUSIONS: Our results suggest that analysis of the dnaJ sequence may be a useful method for the identification of clinical isolates of Vibrio species, especially for distinguishing between closely related Vibrio species.
Subject(s)
Humans , Cholera , Diagnostic Errors , DNA, Ribosomal , Fatal Outcome , Methods , Polymerase Chain Reaction , Sequence Analysis , VibrioABSTRACT
Background: Loop-mediated isothermal amplification (LAMP) is a method already claimed as a simple technique to amplify DNA/ RNA using four to six primers as “a set” from conserved sequence of target gene. In this study we optimize the use of LAMP for detection of Mycobacterium tuberculosis in clinical isolates from Indonesia. Methods: Procedures to perform LAMP were optimized, then the method was applied to 122 archieved samples of DNA’s Mtb from clinical TB patients with Acid Fast Bacilli (AFB) smears positive. The samples were obtained in 2008 from 13 provinces in Indonesia for genotyping study, which then become collections of Center for Biomedical and Basic Technology of Health (CBBTH), NIHRD Indonesia. The optimization tests include sensitivity and specificity tests of several sets primers, which were evaluated using 10-fold serially diluted DNA of Mtb H37Rv and 12 species of Mycobacteria. Three equipments consisted of LAMP turbidimeter, heating block and water bath were compared for its ability in DNA amplification. Detection of M. tuberculosis from clinical isolates used set primers specific for gyrB gene, amplicon was detected with UV fluorescence system. Results: The results showed that the highest sensitivity was obtained using the set primers specific for 16S rRNA and gyrB which could detect 10.0 fg to 1.0 pg genomic DNA of Mtb H37Rv. The set primers specific for gyrB gene was the most specific primers. Application of LAMP using gyrB set primers on Indonesian clinical isolates showed 94.2% (114/121) positivity rate. Conclusion: LAMP method is potentially used in TB diagnosis in Indonesia.
Subject(s)
Mycobacterium tuberculosisABSTRACT
Background: A progressive frequency of resistance to fluorquinolones is observed among Gram-negative bacilli. Aim: To investigate the mechanism of resistance to fluoroquinolones mediated by mutations affecting gyrA and gyrB genes in strains of Gram negative bacüli isolated from CMean hospitals. Material and method: Minimal inhibitory concentration of fluoroquinolones was determined in 91 randomly selected nalidixic acid-resistant strains of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, isolated from hospitals of 12 Chilean cities. Quinolone resistance determining region (QRDR) was amplified by PCR and mutations were determined by restriction fragment length polymorphism (RFLP) and DNA sequencing. Results: Strains with mutation in codon 83 of gyrA showed decreased susceptibility to ciprofloxacin with MICs ranging from 0.25 to 1024 fig/ml. The sequencing of PCR products for gyrA indicated amino acid changes in the QRDR region. One strain ofE. coli presented a double mutation, in codon 83 Ser to Leu as well as in codon 87 Asp to Asn. In strains ofK. pneumoniae, however, the change of codon 83 was Ser to Tyr, in A. baumannii was Ser to Leu and in P. aeruginosa was Thr to He. No strains with mutations affecting gyrB were found. Conclusions: Mutations in codon 83 of gyrA is a frequent genetic event involved in the mechanism leading to decreased susceptibility to fluoroquinolone in strains of Gram-negative bacilli.