[Objective]To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida(PM),Haomophilus parasuis(HPS)and Actinbaci/lus pleuropneumoniae(App).[Method]PCR method was developed to detect sing...[Objective]To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida(PM),Haomophilus parasuis(HPS)and Actinbaci/lus pleuropneumoniae(App).[Method]PCR method was developed to detect single infection caused by PM,HPS or App.The conditions of amplification and primers were optimized,and the multiple-PCR was developed to detect mixed infection of PM,HPS and App.[Result]A 457-bp band,a 821-bp band and a 342-bp band were simultaneously amplified in the one PCR reaction system.The method had high sensitivity and specificity.[Conduslon]The multiple-PCR is successfully developed and can be used for differential diagnosis of PM,HPS and App.展开更多
The use of an internal control in a multiplex-PCR assay for sex determination of In Vitro-produced bovine embryos was evaluated in biopsies of groups of 54 fresh and 44 frozen embryos. The internal controls used were ...The use of an internal control in a multiplex-PCR assay for sex determination of In Vitro-produced bovine embryos was evaluated in biopsies of groups of 54 fresh and 44 frozen embryos. The internal controls used were the primers BOV 1 and BOV 2, which amplify a product with 626 base pairs (bp) of bovine mitochondrial DNA ND5 gene. The primers BRY.4aF and BRY.4aR were used for bovine Y chromosome sequence amplification. The specificity of multiplex- PCR reactions realized in biopsies corresponding to about 20% of each fresh embryo (10 male and 10 female) by means of confirming the sexing in the remaining embryo content (~80%) presented 100% specificity. Amplicons of the internal control and Y chromosome were both amplified until dilution corresponding to 6.25% of total extracted DNA from a male embryo. Sex determination was possible in 53 (98.1%) fresh embryos and 40 (90.9%) frozen embryos. The products related to the Y chromosome and mitochondrial DNA were simultaneously amplified in 34 (63%) fresh embryos and 27 (61.4%) frozen embryos, showing a male embryo. The female sex, distinguished by internal control amplification only, was detected in 19 (35.2%) and 13 (29.5%) biopsies, respectively, of fresh and frozen embryos. In one (1.8%) and four (9.1%) biopsies of fresh and frozen embryos, respectively, neither product was amplified, most likely due to the absence of embryonic cells or the presence of embryonic cells going through apoptosis. The multiplex-PCR assay developed in this work showed avoided the limitation of a lack of an internal standard, and was also sensitive, specific, and efficient in reaction failure identification. This technique shows great potential for use on a commercial scale in routine sex determination of In Vitro-produced embryos.展开更多
目的探讨广东清远地区女性患者人乳头瘤病毒(HPV)感染现状和亚型分布特点,为HPV感染防治和疫苗接种提供临床依据。方法收集清远市人民医院2021年1月至2022年12月HPV DNA 25分型检测的16926例女性患者数据,回顾性分析患者HPV基因型分布...目的探讨广东清远地区女性患者人乳头瘤病毒(HPV)感染现状和亚型分布特点,为HPV感染防治和疫苗接种提供临床依据。方法收集清远市人民医院2021年1月至2022年12月HPV DNA 25分型检测的16926例女性患者数据,回顾性分析患者HPV基因型分布和不同年龄层、不同宫颈病变程度HPV感染特点。结果本研究共纳入数据16926例,HPV阳性感染3563例,阳性率为21.05%,高危型检出率前5位分别是HPV52、58、16、39和51,低危型最常见型别为HPV42、81和6。分析女性患者各年龄层组间差异发现11~20岁HPV阳性检出率最高(42.38%),HPV感染随着年龄的增加呈现先下降后升高的趋势。不同程度宫颈病变患者感染型别最多依次为HPV52、58和16,HPV感染随着宫颈病变程度的加重呈现出先升高再下降的趋势。各宫颈病变组HPV单一感染占比(23.89%)明显高于多重感染(10.02%),单一和多重感染中均以高危型HPV感染为主,且差异有统计学意义(P均<0.05)。结论清远地区女性HPV感染以HPV52、58、16、39和51型为主,11~20岁和>60岁患者阳性率较高,不同程度宫颈病变患者多见单一高危型感染。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
基金funded by the subproject of National Key Technology R&D Program(2006BAD06A01 and 2006BAD06A12)Basic Work of National Science and Technology Special Plan(2008FY210200)
文摘[Objective]To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida(PM),Haomophilus parasuis(HPS)and Actinbaci/lus pleuropneumoniae(App).[Method]PCR method was developed to detect single infection caused by PM,HPS or App.The conditions of amplification and primers were optimized,and the multiple-PCR was developed to detect mixed infection of PM,HPS and App.[Result]A 457-bp band,a 821-bp band and a 342-bp band were simultaneously amplified in the one PCR reaction system.The method had high sensitivity and specificity.[Conduslon]The multiple-PCR is successfully developed and can be used for differential diagnosis of PM,HPS and App.
文摘The use of an internal control in a multiplex-PCR assay for sex determination of In Vitro-produced bovine embryos was evaluated in biopsies of groups of 54 fresh and 44 frozen embryos. The internal controls used were the primers BOV 1 and BOV 2, which amplify a product with 626 base pairs (bp) of bovine mitochondrial DNA ND5 gene. The primers BRY.4aF and BRY.4aR were used for bovine Y chromosome sequence amplification. The specificity of multiplex- PCR reactions realized in biopsies corresponding to about 20% of each fresh embryo (10 male and 10 female) by means of confirming the sexing in the remaining embryo content (~80%) presented 100% specificity. Amplicons of the internal control and Y chromosome were both amplified until dilution corresponding to 6.25% of total extracted DNA from a male embryo. Sex determination was possible in 53 (98.1%) fresh embryos and 40 (90.9%) frozen embryos. The products related to the Y chromosome and mitochondrial DNA were simultaneously amplified in 34 (63%) fresh embryos and 27 (61.4%) frozen embryos, showing a male embryo. The female sex, distinguished by internal control amplification only, was detected in 19 (35.2%) and 13 (29.5%) biopsies, respectively, of fresh and frozen embryos. In one (1.8%) and four (9.1%) biopsies of fresh and frozen embryos, respectively, neither product was amplified, most likely due to the absence of embryonic cells or the presence of embryonic cells going through apoptosis. The multiplex-PCR assay developed in this work showed avoided the limitation of a lack of an internal standard, and was also sensitive, specific, and efficient in reaction failure identification. This technique shows great potential for use on a commercial scale in routine sex determination of In Vitro-produced embryos.
文摘目的探讨广东清远地区女性患者人乳头瘤病毒(HPV)感染现状和亚型分布特点,为HPV感染防治和疫苗接种提供临床依据。方法收集清远市人民医院2021年1月至2022年12月HPV DNA 25分型检测的16926例女性患者数据,回顾性分析患者HPV基因型分布和不同年龄层、不同宫颈病变程度HPV感染特点。结果本研究共纳入数据16926例,HPV阳性感染3563例,阳性率为21.05%,高危型检出率前5位分别是HPV52、58、16、39和51,低危型最常见型别为HPV42、81和6。分析女性患者各年龄层组间差异发现11~20岁HPV阳性检出率最高(42.38%),HPV感染随着年龄的增加呈现先下降后升高的趋势。不同程度宫颈病变患者感染型别最多依次为HPV52、58和16,HPV感染随着宫颈病变程度的加重呈现出先升高再下降的趋势。各宫颈病变组HPV单一感染占比(23.89%)明显高于多重感染(10.02%),单一和多重感染中均以高危型HPV感染为主,且差异有统计学意义(P均<0.05)。结论清远地区女性HPV感染以HPV52、58、16、39和51型为主,11~20岁和>60岁患者阳性率较高,不同程度宫颈病变患者多见单一高危型感染。
