Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of ...Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.展开更多
Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three t...Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.展开更多
文摘Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.
基金funded by the National Key R&D Program of China[2021YFC2301102]National Natural Science Foundation of China[82202593]Key R&D Program of Hebei Province[223777100D].
文摘Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.
文摘目的本研究旨在建立一种实时荧光定量PCR方法,用于检测猕猴三磷酸腺苷结合盒转运蛋白G2(adenosine triphosphate-binding cassette transporter protein G2,ABCG2)mRNA的基因转录水平。方法使用NCBI上GenBank数据库猕猴(Macaca mulatta)的ABCG2核苷酸序列号NM_001032919.1及内参GAPDH核苷酸序列号NM_001195426.1,借助Primer premier 5.0软件设计PCR引物。提取猕猴新鲜肾组织的总RNA,并反转录合成cDNA。接着,利用PCR引物进行实时荧光定量PCR扩增,并根据反应体系中荧光的变化情况定量分析ABCG2的mRNA相对表达水平。结果PCR产物测序结果显示,扩增的ABCG2和GAPDH核苷酸序列与NCBI上猕猴的序列同源性分别为90.91%和91.14%。ABCG2和GAPDH的扩增效率均达到80%~120%,实时荧光定量PCR标准曲线的熔解曲线为单峰,R2接近1。结论本研究建立的检测猕猴ABCG2 mRNA实时荧光定量检测方法,为研究高尿酸血症的发病机制以及新药开发奠定基础。