Molecular identification of Trichophyton mentagrophytes complex strains in Hubei province / 中华皮肤科杂志

Objective To profile the intraspecific type of Trichophyton mentagrophytes clinically isolated from different anatomical sites of patients, and to compare the performance of different target sites for the identification of Trichophyton mentagrophytes complex strains. Methods A total of 48 Trichophyton mentagrophytes strains, which were clinically isolated from Department of Dermatology, Wuhan No. 1 Hospital in the latest 3 years, were included in this study. The phenotypes of these Trichophyton mentagrophytes isolates were primarily determined by morphological observation and the urease test. PCR was performed to amplify the nuclear ribosomal internal transcribed spacer(ITS) region and the D1?D2 domains of the large?subunit ribosomal DNA(28S rDNA)followed by DNA sequencing. Then, Clustal X2 software and MEGA 6.0 software were used to analyze the ITS and D1?D2 sequences and to build phylogenetic trees by the maximum?likelihood method (bootstrap = 2000). Results As the ITS sequence?based phylogenetic tree showed, the probability that the 48 isolates were grouped into the Trichophyton interdigitale clade reached 92%. However, Trichophyton interdigitale could not be effectively differentiated from Trichophyton quinckeanum by the D1?D2 sequence?based phylogenetic tree. In addition, Trichophyton interdigitale showed various appearances, including woolen type, downy type, cream type, powdery type and granular type. Conclusions Trichophyton mentagrophytes can be identified to the species level based on the sequence of ITS region, which shows higher efficiency in identifying Trichophyton mentagrophytes complex than the D1?D2 domains. Morphological characteristics can not serve as the basis for intraspecific typing of Trichophyton mentagrophytes.

The ability to identify the yeast-like fungi by YST identification cards and pyrosequencing analysis / 中华检验医学杂志

Objective To evaluate the ability of Vitek 2 Compact YST identification cards and pyrosequencing analysis for identifying the clinical isolates of yeast-like fungi.Methods Vitek 2 Compact YST identification cams and ITS1 region pyrosequencing analysis were used to identify the clinical isolates of yeast-like fungi at Jinan Military General Hospital in 2011.The strains which could not be identified to species by pyrosequencing analysis were identified again by the method of ITS1 region Sanger sequencing.The strains with inconsistent identified results by YST and pyrosequencing were identified again using API 20C.Results A total of 282 srtains were isolated from various clinical specimens for culturing in 2011.In addition to the three which could only be identified to similar species,other strains could be clearly identified to species by the method of ITS1 reverse pyrosequencing.Three strains which failed identified to species by pyrosequencing couldn't be distinguished fully by the method of Sanger sequencing.There were 7 strains unidentified,14 strains low discrimination(％ id ＜ 80％),and 6 strains misidentification using YST cams.The identification coincidence rate was 90.4％.The identification coincidence rates were various in different species of yeast-like fungi using YST cards.Conclusions The vast majority of clinical isolates of yeast-like fungi could be identified by ITS1 reverse pyrosequencing analysis.Vitek 2 Compact YST caRD basically meets the needs.But attention should be paid to the standard operating and regular internal quality control.For some rare species it is also needed to combine the source of specimen,colonies and characteristics to determine the identification results for the rare stains.

The clinical application of identification of Aspergillus spp.using ITS and β-tubulin gene sequences / 中华检验医学杂志

Objective To study the clinical application of the ITS and β-tubulin gene regions in identification of Aspergillus spp. Methods One hundred and twenty-four Aspergillus strains that isolated from fungal rhino-sinusitis specimens were collected in Beijing Tongren Hospital, Capital Medical University from July 2007 to January 2010. They were identified by morphological and molecular methods. The first one included traditional culture, slide culture, and microscopic examination after lactophenol cotton blue stain and KOH digestion. The second one was amplifying and sequencing the part of ITS and β-tubulin gene and aligned all the sequences in the GenBank, European Molecular Biology Laboratory nucleotide sequence database, and DNA Data Bank of Japan. Results Of the 56 Aspergillus flavus identified by morphological features, fifty-five isolates were identified as Aspergillus flavus and 1 isolates was Aspergillus parasiticus by the ITS and β-tubulin gene region sequence analysis. In the 37 Aspergillus fumigatus identified by morphological method, and all the 37 isolates were identified as species complex of Aspergillus fumigatus by the ITS region sequence analysis, but through the sequence analysis of β-tubulin gene region, thirty-five isolates were identified as Aspergillus. fumigatus and 2 were Aspergillus lentulus. Twenty-one isolates were identified as Aspergillus versicolor by morphological method, but 16 of them were identified as Aspergillus. versicolor and 5 can not be identified to species level by the ITS region sequence. And by comparative-sequence analysis of β-tubulin gene region, the 5 isolates were identified as Aspergillus sydowii,the other 16 isolates were Aspergillus. versilcolor. Ten isolates were identified as Aspergillus nidulans by morphological features, the ITS and β-tubulin gene region sequence analysis. Conclusions β-tubulin gene sequencing is more suitable for identifying Aspergillus, and could identify Aspergillus spp. to species level Sequences of ITS region could only identify Aspergillus spp. to species complex.

A rapid molecular taxonomy technique on the bacteria genus and species by 16S rDNA and 16S-23S rDNA intergenic spacer regions sequences / 中华检验医学杂志

Objective To explore a rapid method for classification of microorganisms.Methods The electrophorese fingerprinting, direct sequence of 16S rDNA and 16S-23S rDNA ISR after PCR, multiplex PCR for 16S rDNA and antibiotic resistance genes, were utilized to explore fast approaches of extracting total DNA from different clinical specimens.Results The specific 16S-23S rDNA ISR fingerprinting fragments were shown on the genus or species level in bacteria and fungi.So fingerprinting can be used to identify pathogenic microorganisms, to differentiate the evolution relations or to set the phylogenetic tree by comparing their DNA banding patterns with those of standard strains (NCCLS). Multiplex PCR was able to examine the special genes of genus or species, mecA gene, TEM, SHV and CTX gene in staphylococcus and ESBLs(E.coli or K.pneumoniae) at the same time.Conclusion The part of 16S rDNA sequencing and 16S-23S rDNA ISR genotypes by gel electrophoreses were useful for bacterial species identification in addition, it was clearly more rapid and accurate than culture technique, and the large numbers of strains can easily be examined.Multiplex PCR could provide a good method for identification of microorganisms and analysis of antibiotic resistance at the same time.

Identification of Sporothrix schenckii Based on the Nucleotide Sequences in Internal Transcribed Spacer Region 2 of Ribosomal DNA Genes / 中华皮肤科杂志

Objective To study the molecular identification method of Sporothrix schenckii based on the nucleotide sequences in internal transcribed spacer region 2 of ribosomal DNA (rDNA) genes. Methods Species-specific primers were used to amplify the internal transcribed spacer region 2 of rDNA of 22 clinical isolates of Sporothrix schenckii and 12 strains of dematiaceous fungi. Totally 11 strains of Sporothrix schenckii were sequenced and analyzed, in which 1 strain came from the US and the others were isolated from different areas in China. A pair of species-specific oligonucleotide primers (SSP) were designed based on the ITS2 sequence. With the species-specific primers, rDNA of Sporothrix schenckii and dematiaceous fungi were amplified by PCR. Results Sequencing and analysis showed that internal transcribed spacer region 2 of rDNA gene was conservative in Sporothrix schenckii. A 300 bp fragment was obtained from 22 strains of Sporothrix schenckii, but not from the other species. Conclusions This method is specific, sensitive and reliable for the identification of Sporothrix schenckii and could be used for clinical molecular diagnosis.