[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli （STEC）. [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR ...[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli （STEC）. [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR primers and probes were designed based on the conserved region to construct recombinant plasmid as a positive template, thus optimizing the reaction conditions and establishing the real- time PCR method. [Result] A standard curve was established based on the opti- mized real-time PCR system, indicting a good linear correlation between the initial template concentration and Ct value, with the correlation coefficient F^e of above 0.995. The established method had a good specificity, without non-specific amplifica- tion for 10 non-STEC intestinal bacterial strains; the detection limit of initial template was 1.0x102 copies/μI, indicating a high sensitivity; furthermore, the coefficients of variation within and among batches were lower than 1% and 5% respectively, sug- gesting a good repeatability. [Conclusion] In this study, a real-time PCR method was successfully established for detecting STEC stx2 gene, which provided technical means for rapid detection of STEC in samples.展开更多
Binary toxin(CDT)-producing Clostridioides difficile is associated with severe clinical symptoms and high mortality rates.However,the fundamental genomic factors that underpin the differentiated prevalence of CDT-prod...Binary toxin(CDT)-producing Clostridioides difficile is associated with severe clinical symptoms and high mortality rates.However,the fundamental genomic factors that underpin the differentiated prevalence of CDT-producing C.difficile strains in China remain to be elucidated.To provide a comprehensive view of the genetic characteristics,this study comprehensively analyzed whole-genome sequences of CDT-producing C.difficile strains from China.The number of unique accessory genes in sequence type(ST)-5 strains,which are predominant in China,increased steadily with the increasing number of genomes sampled,while the accumulation curve for ST-1 strains was shallower.This suggests that ST-5 may have an open pangenome,whereas ST-1 may have a relatively conservative and stable pangenome.The pathogenicity locus(PaLoc)variants of Clades 1,2 and 5 were found to share a common genetic organization,with the CDT locus(CdtLoc)showing lower variability than PaLoc.PaLoc in Clade 3 exhibits an insertion of transposon Tn6218,which is clade-specific.The flexibility of the C.difficile accessory genome has facilitated the development of divergent lineages characterized by the presence of evolutionarily advantageous genetic traits.This genetic diversification has led to enhanced antimicrobial resistance,increased virulence and improved metabolic capacities,which collectively augment the ability of C.difficile to survive and proliferate.展开更多
基金Supported by Agricultural Science and Technology Support Program(Social Development)of Jiangsu Province(BE2011771)~~
文摘[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli （STEC）. [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR primers and probes were designed based on the conserved region to construct recombinant plasmid as a positive template, thus optimizing the reaction conditions and establishing the real- time PCR method. [Result] A standard curve was established based on the opti- mized real-time PCR system, indicting a good linear correlation between the initial template concentration and Ct value, with the correlation coefficient F^e of above 0.995. The established method had a good specificity, without non-specific amplifica- tion for 10 non-STEC intestinal bacterial strains; the detection limit of initial template was 1.0x102 copies/μI, indicating a high sensitivity; furthermore, the coefficients of variation within and among batches were lower than 1% and 5% respectively, sug- gesting a good repeatability. [Conclusion] In this study, a real-time PCR method was successfully established for detecting STEC stx2 gene, which provided technical means for rapid detection of STEC in samples.
基金supported by the Basic Public Welfare Research Program of Zhejiang Province(LGC20C010002)the National Nature Science Foundation of China(82073609)and Shandong Provincial Laboratory Project(SYS202202)。
文摘Binary toxin(CDT)-producing Clostridioides difficile is associated with severe clinical symptoms and high mortality rates.However,the fundamental genomic factors that underpin the differentiated prevalence of CDT-producing C.difficile strains in China remain to be elucidated.To provide a comprehensive view of the genetic characteristics,this study comprehensively analyzed whole-genome sequences of CDT-producing C.difficile strains from China.The number of unique accessory genes in sequence type(ST)-5 strains,which are predominant in China,increased steadily with the increasing number of genomes sampled,while the accumulation curve for ST-1 strains was shallower.This suggests that ST-5 may have an open pangenome,whereas ST-1 may have a relatively conservative and stable pangenome.The pathogenicity locus(PaLoc)variants of Clades 1,2 and 5 were found to share a common genetic organization,with the CDT locus(CdtLoc)showing lower variability than PaLoc.PaLoc in Clade 3 exhibits an insertion of transposon Tn6218,which is clade-specific.The flexibility of the C.difficile accessory genome has facilitated the development of divergent lineages characterized by the presence of evolutionarily advantageous genetic traits.This genetic diversification has led to enhanced antimicrobial resistance,increased virulence and improved metabolic capacities,which collectively augment the ability of C.difficile to survive and proliferate.
