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
仙人掌X病毒(cactuSviruSX,CVX)严重威胁世界火龙果产业的发展。为实现对该病毒的定量检测,本研究根据CVX外壳蛋白(CP)的保守序列设计特异性引物CVX-F/-R,建立了引物浓度150 nmol/L,退火温度62℃的SYBR Green I实时荧光定量PCR检测方法...仙人掌X病毒(cactuSviruSX,CVX)严重威胁世界火龙果产业的发展。为实现对该病毒的定量检测,本研究根据CVX外壳蛋白(CP)的保守序列设计特异性引物CVX-F/-R,建立了引物浓度150 nmol/L,退火温度62℃的SYBR Green I实时荧光定量PCR检测方法。该引物扩增效率为97.38%,R^(2)为0.9965,对标准品的检测极限浓度为8×10^(2)拷贝/μL,其灵敏度是普通PCR的100倍。对火龙果不同组织中CVX的相对表达量进行检测发现,病毒在花丝中的含量最高,之后依次为花萼、花瓣、嫩枝、老枝和根。对采自贵州黔南州的40份火龙果样品进行检测,共检测出32份阳性样品。上述结果表明,本研究建立的实时荧光定量PCR特异性强、灵敏度高,为今后CVX的检测提供了重要的技术补充。展开更多
Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and ...Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and resulting in huge economic losses. Using PCR technology has very important significance to the correct diagnosis of Mycoplasmal pneumonia at the early stage. In this paper, specific target genes of Mycoplasma hyopneumoniae, methods for clinical sample collection, key technical factors of DNA sample processing method, and the research progress, main advantages and disadvantages, and application of general PCR technology, multiple PCR technology, nested-PCR technology, real-time fluorescence quantitative PCR technology, gene chip detection technology and loop-mediated isothermal amplification in detection of Mycoplasma hyopneumoniae were summarized, which provided convenience for the effective diagnosis and prevention of Mycoplasmal pneumonia of swine.展开更多
为了满足乳制品中食源性致病菌精确、高效和高通量检测需求,文章以典型食源性致病菌大肠埃希氏菌O157、金黄色葡萄球菌、单核细胞增生李斯特氏菌和沙门氏菌作为目的菌,建立1种灵敏稳定的多重芯片式数字PCR(Multiplex digital chip PCR)...为了满足乳制品中食源性致病菌精确、高效和高通量检测需求,文章以典型食源性致病菌大肠埃希氏菌O157、金黄色葡萄球菌、单核细胞增生李斯特氏菌和沙门氏菌作为目的菌,建立1种灵敏稳定的多重芯片式数字PCR(Multiplex digital chip PCR)反应体系。针对4种致病菌的靶向基因,设计特异性引物,优化多重反应引物组合和反应条件,并验证其特异性和灵敏度。结果表明,该四重反应体系具有良好的特异性且未出现交叉反应。体系对4种菌株最低检出限分别达0.38、1.03、0.69 copies/μL和0.24 copies/μL,与单重反应体系检测灵敏度相当,未发生混合引物抑制反应。方法对模拟阳性样品、生乳和市售乳制品检测结果证明了其在乳原料及乳制品质量安全控制中的应用前景,文章构建的四重反应体系可为乳制品的检测与监管提供技术支持。展开更多
We developed one species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Verticillium albo-atrum in diseased plant tissues and soil. Based on differences in internal transcribed spacer （...We developed one species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Verticillium albo-atrum in diseased plant tissues and soil. Based on differences in internal transcribed spacer （ITS） sequences of Verticillium spp., a pair of species-specific primers, Vaal/Vaa2, was synthesized. After screening 17 isolates of V. alboatrum, 121 isolates from the Ascomycota, B asidiomycota, Deuteromycota, and Oomycota, the Vaal/Vaa2 primers amplified only a single PCR band of approximately 330 bp from V. albo-atrum. The detection sensitivity with primers Vaal/Vaa2 was 10 fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with Vaa1/Vaa2, the nested PCR procedures were developed, and the detection sensitivity increased 1 000-fold to 10 ag. The detection sensitivity for the soil pathogens was 100-conidiag^-1 soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.展开更多
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.
文摘仙人掌X病毒(cactuSviruSX,CVX)严重威胁世界火龙果产业的发展。为实现对该病毒的定量检测,本研究根据CVX外壳蛋白(CP)的保守序列设计特异性引物CVX-F/-R,建立了引物浓度150 nmol/L,退火温度62℃的SYBR Green I实时荧光定量PCR检测方法。该引物扩增效率为97.38%,R^(2)为0.9965,对标准品的检测极限浓度为8×10^(2)拷贝/μL,其灵敏度是普通PCR的100倍。对火龙果不同组织中CVX的相对表达量进行检测发现,病毒在花丝中的含量最高,之后依次为花萼、花瓣、嫩枝、老枝和根。对采自贵州黔南州的40份火龙果样品进行检测,共检测出32份阳性样品。上述结果表明,本研究建立的实时荧光定量PCR特异性强、灵敏度高,为今后CVX的检测提供了重要的技术补充。
基金Supported by National Natural Science Foundation of China(31100135)Agricultural Independent Innovation Fund of Jiangsu Province[CX(11)4038]~~
文摘Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and resulting in huge economic losses. Using PCR technology has very important significance to the correct diagnosis of Mycoplasmal pneumonia at the early stage. In this paper, specific target genes of Mycoplasma hyopneumoniae, methods for clinical sample collection, key technical factors of DNA sample processing method, and the research progress, main advantages and disadvantages, and application of general PCR technology, multiple PCR technology, nested-PCR technology, real-time fluorescence quantitative PCR technology, gene chip detection technology and loop-mediated isothermal amplification in detection of Mycoplasma hyopneumoniae were summarized, which provided convenience for the effective diagnosis and prevention of Mycoplasmal pneumonia of swine.
基金We thank Dr Chen Qinghe,Dr Ko Wenhong,Dr Ho Hanhin,Dr Hu Baishi,Dr Peng Jinghuo and China General Microbiological Culture Collection Center(CGMCC)for providing some isolates.This work was supported by the China National 863 Program(2003AA249020).
文摘We developed one species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Verticillium albo-atrum in diseased plant tissues and soil. Based on differences in internal transcribed spacer （ITS） sequences of Verticillium spp., a pair of species-specific primers, Vaal/Vaa2, was synthesized. After screening 17 isolates of V. alboatrum, 121 isolates from the Ascomycota, B asidiomycota, Deuteromycota, and Oomycota, the Vaal/Vaa2 primers amplified only a single PCR band of approximately 330 bp from V. albo-atrum. The detection sensitivity with primers Vaal/Vaa2 was 10 fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with Vaa1/Vaa2, the nested PCR procedures were developed, and the detection sensitivity increased 1 000-fold to 10 ag. The detection sensitivity for the soil pathogens was 100-conidiag^-1 soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.
