Application of OrthoANI combined with MALDI-TOF MS in identification and typing of Campylobacter fetus / 中华检验医学杂志

Objective@#Establishing the mass spectrum library of a new Campylobacter- " C.fetus subsp.testudinum" for rapid species identification in clinical microbiology laboratory.@*Methods@#Illumina second generation sequencing platform 2000/miSeq was used to carry out high flux genome sequencing for the strains which were collected to establish mass spectrum library.The analysis oforthologous average nucleotide identity (OrthoANI) between collected strains and reference strains was performed at JAVA 8 operation environment. Then, the mass spectrums ofcollected strains andreference strains were acquired using MALDI-TOF MS. And the mass spectrum library of C. fetus subsp.testudinum. were established and verified.@*Results@#The OrthoANI analysis showed that the OrthoANI value of the collected strains and the reference strain C. fetus subsp.testudinum03-427 was 99.30%-99.96%, while the OrthoANI values of collected strains and C. fetus subsp.venerealisNCTC10354 orC.fetus subsp.fetus82-40 were 91.05%-92.26%. With reference to OrthoANI ≥ 95% as the basis for the determination of the same strain, the strains which collected to establish mass spectrum library was finally identified as " C. fetus subsp.testudinum" . The identification accuracy rate of the mass spectrum library was 100% (consistent with gene sequencing), and the confidence interval was 82.3%-99.9%, identification of the same strain is 100% reproducible.@*Conclusions@#The new" gold standard" based on high throughput sequencing and total genome analysis has provided the ideal reference value for the establishment of mass spectrum library.And the accurate and objective reference spectrum of the" C.fetus subsp.testudinum" provides a new platform for the rapid diagnosis of fetal Campylobacter infection. (Chin J Lab Med, 2018, 41: 583-588)

Identification of a newly reported Francisella species by average nucleotide identity based on high-throughput whole genome sequencing technology / 临床检验杂志

Objectives To identify the Francisella strain isolated from blood of a patient with drowning-associated pneumonia.Methods The whole genome of the strain,designated Wenzhou1,was sequenced using the high throughput sequencing technology by 2000/miSeq system of Illumina platform,and the obtained genome draft was assembled by MicrobeTrakr Plus software.The phylogenetic neighbors of Wenzhou1 were obtained by NCBI BLAST analysis from GenBank database for the gene sequences of 16S rRNA,malate dehydrogenase(mdh),DNA-directed RNA polymerase subunit beta (rpoB) and succinate dehydrogenase subunit alpha (sdhA).The average nucleotide identity(ANI) between Wenzhou1 and its phylogenetic neighbors was analyzed by the software OrthoANI using NCBI BLAST search under the Java Runtime Environment Version 8.Results The genome size of Wenzhou1 was 1.96 × 106 bp,containing 74 contigs.The genomic G + C mol％ of Wenzhou1 was 32.1％,which was similar to the other species of genus Francisella and Allofranicella.Based on the analysis of NCBI BLAST of GenBank for the similarities of 16S rRNA gene,mdh gene,rpoB gene and sdbA gene sequences,Wenzhou1 was most closely related to F.hispaniensis FSC454 and Francisella cf.novicida 3523.The ANI of Wenzhou1 was 97.8％ to F.hispaniensis FSC454,97.5％ to 97.6％ to Francisella cf.novicida 3523,but only 91.3％ to 91.5％ to the four subspecies of F.tularensis.Conclusion ANI analysis based on whole genome sequence should be an accurate,effective method for bacterial identification.Wenzhou1 could be identified as F.hispaniensis by ANI with high-throughput whole genome sequencing technology.

Influence of Gleevec on expression of miR-146a,miR-29b and DNMT in K562 cells / 重庆医学

Objective To investigate the changes of microRNA (miR)-146a ,miR-29b expression levels and the 3 kinds of meth-ylase DNMT1 ,DNMT3a and DNMT3b levels in K562 cell lines after BCR/ABL inhibitor Gleevec treatment .Methods The half maximal inhibitory concentration(IC50 ) of Gleevec on K562 cells was detected by the MTT method .The stem loop primers method and the fluorogenic quantitative PCR were adopted to detect miRNAs and the methylase gene level .Results IC50 of Gleevec acting on K562 cells was 40 .85μmol/L .After Gleevec action ,miR-29b showed the increasing trend ,but 3 kinds of methylase expression level were decreased to some extent .Gleevec could significantly increase the miR-146a level in K562 cells(P<0 .05) .Conclusion Gleevec can influence the expression of miR-146a ,miR-29b and DNMTs levels in K562 cells .

Use ITS gene sequence analysis to identify dematiaceous fungi / 实用医学杂志

Objectives Use ITS gene sequence analysis to identify 15 strains of dematiaceous fungi , to learn the types of pathogenic strains and clinical treatment. Methods By observing the colony morphology and microscope morphological of the dematiaceous fungi isolated from superficial mycoses , and identified by ITS gene sequence analysis. Results 15 strains were identified by morphological observation as dematiaceous fungi.The amplified bands were identified by Tanon-3500 gel imaging system between 500 ~ 700 bp. Blast sequencing results show that 2 strains Alternaria alternate , 2 strains Cladosporium sphaerospermum. 2 strains Exophiala dermatitis, 1 strains Cladosporium cladosporioides, Curvularia lunata, Talaromyces rugulosus, Phaeobotryon cupressi, Cladosporium tenuissimum, Fonseceea pedrosoi, Exophiala werneckii, Exophiala oligosperma and Fonsecaea monophora. Conclusion ITS gene sequence analysis can identify dematiaceous fungi effectively , avoided undetected and misdiagnose cause by the lack of clinical experience.

Identification of Streptobacillus moniliformis isolated from the knee joint pus by 16S rRNA gene sequencing and biochemical reactions / 实用医学杂志

Objective To indentify Streptobacillus moniliformis isolated from the knee joint pus by 16S rRNA gene sequencing and biochemical reactions and explore the clinical value of the method. Methods The bacterial 16S rRNA gene sequence-based identification, bacterial morphology, VITEK 2 automate systems, API 20NE strips, API 20E strips and API 50CH were performed to identify the rare bacteria. Results The bacteria grew slow on blood agar and chocolate agar and were inhibited on Maconkey agar. The bacterial colony on blood agar tookes the form of 1~2 mmomelette, which was translucent and moist with circular protrusion and smooth edges. They were Gram-staining negative and in catenation, its thalli 1~3μm, round, oval or fusiform. Vitek 2 GN-13, API 20NE and API 20E were unable to reach the identification of the bacteria. 16S rRNA gene sequencing showed the bacteria were similar to streptobacillus moniliformis by 100%. Conclusion The rare bacteria isolated from left knee joint are streptobacillus moniliformis. 16S rRNA gene sequences combined with the biochemical reactions is accurate in the identification of these bacteria.