Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequence...Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.展开更多
Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfl...Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.展开更多
基金supported by a grant from Beijing Municipal Natural Science Foundation [L212011]National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention [131031102000210003&102393230020020000002]。
文摘Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.
基金supported by the National Natural Science Foundation of China [Grant agreement 31502124]the National Science and Technology Major Project of China [Grant agreement 2018ZX10733402]
文摘Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.
