目的为了提高微量样本中miRNA的检测通量和检测效率,本文建立了一种能够同时准确定量两种miRNA的双重实时荧光定量PCR(real time fluorogenic quantitative PCR)检测体系,并通过实际样本检测验证其应用于体液鉴别的效果。方法设计适用于...目的为了提高微量样本中miRNA的检测通量和检测效率,本文建立了一种能够同时准确定量两种miRNA的双重实时荧光定量PCR(real time fluorogenic quantitative PCR)检测体系,并通过实际样本检测验证其应用于体液鉴别的效果。方法设计适用于miRNA双重检验的相关引物及探针并优化实验体系组分,建立基于TaqMan技术的miRNA双重实时荧光定量PCR检测体系并验证其特异性、灵敏度和可重复性;使用此检测体系对58份不同体液样本中的miR-451a与miR-21-5p进行检测,并借助miR-451a与miR-21-5p的比值鉴定法评估该体系的体液鉴别能力;使用该检测体系样本数据确定的最佳截断值对模拟案件样本进行鉴别。结果优化的检测体系能够实现对血液与非血液、月经血与外周血的100%区分,同时可以实现对模拟案件样本的准确鉴别。结论该双重实时荧光定量PCR检测体系将时间和材料成本均缩短至原来的一半,为后续建立更多重的实时荧光定量PCR检测体系并应用于体液鉴别打下了基础。展开更多
为建立一种能快速对羊泰勒虫(ovine and caprine theileria)和嗜吞噬细胞无浆体(A.phagocytoph-ilum)同时进行检测的双重PCR方法。根据GenBank已报道的羊泰勒虫MPSP基因和羊嗜吞噬细胞无浆体16S rRNA基因设计合成了2对特异性引物,通过...为建立一种能快速对羊泰勒虫(ovine and caprine theileria)和嗜吞噬细胞无浆体(A.phagocytoph-ilum)同时进行检测的双重PCR方法。根据GenBank已报道的羊泰勒虫MPSP基因和羊嗜吞噬细胞无浆体16S rRNA基因设计合成了2对特异性引物,通过条件优化,建立了双重PCR检测方法。双重PCR可特异扩增出羊泰勒虫和嗜吞噬细胞无浆体目的条带,片段大小分别为875bp和394bp。该方法具有较好的特异性,对羊泰勒虫和嗜吞噬细胞无浆体的最低检出浓度为16fg/μL和1fg/μL。对采集的60份血液样本进行双重PCR检测,羊泰勒虫阳性率为46.67%(28/60),嗜吞噬细胞无浆体阳性率为13.33%(8/60),混合感染率为10%(6/60)。结果表明,双重PCR方法可用于羊泰勒虫和嗜吞噬细胞无浆体的快速诊断和流行病学调查。展开更多
Objective:To identify members of genera of rickettsia and O.tsutsugammhi simultaneously.Methods:Rapid and duplex and nested PCR methods have been established by designing primers based on the conserved regions of heat...Objective:To identify members of genera of rickettsia and O.tsutsugammhi simultaneously.Methods:Rapid and duplex and nested PCR methods have been established by designing primers based on the conserved regions of heat shock protein GroEL gene.345 mouse viscera samples including liver,spleen and kidney,96 Xenopsylla cheopis and 32 chiggers collected from Hongta areas of Yuxi city,Yunnan province were tested by the new PCR methods.Results:The result of the study showed that the new PCR methods could identify most members of genera -Rickettsia and Orientia simultaneously with 100%specificity and its sensitivity could test one copy per microliter.The results of detection prevalence of rickettsioses in mouse,flea and mites DNA samples showed that the total rickettsia infection rate in mouse was 34.78%(120/345).The total infection rates in R.typhi,O.t Karp and R.sibirica of mouse samples were 28.12%(97/345),19.71%(68/345) and O. 29%(1/345) respectively.Co-infection rates in R.typhi and 0.t Karp of mouse samples were 13.33%(46/ 345).O.t Karp type has been the main epidemic strain in these areas.Conclusion:We concluded that this PCR method could be used to detect multi-genera rickettsia simultaneously.Molecular evidences provided in this and previous studies strongly support that Hongta areas of Yuxi city are a natural focus for typhus and scrub typhus with the common occurrence of their confection.展开更多
Objective:To design a duplex PCR for rapid and simultaneous detection of Brucella species, in human blood samples.Methods:Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis.Fo...Objective:To design a duplex PCR for rapid and simultaneous detection of Brucella species, in human blood samples.Methods:Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis.Following DNA extraction,PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals.Results:Of the 52 peripheral bloods samples tested, 25 sample(48%) showed positive reactions in PCR.Twelve samples were positive for Brucella abortus(B.abortus)(23%,13 for Brucella melUensis(B.melUensis)(25%) and 0 for Brucella ovis (6.ovis)(Ow.Conclusions:This work de=monstrates dial in case where specific primers were utilized,duplex PCR has proved to be a simple,fast,and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples.展开更多
H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg producti...H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction（RT-PCR） assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR（d RT-PCR） was established. Two primer sets target the hemagglutinin（HA） gene of H9 AIV and the nucleocapsid（N） gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10^1, 1.5×10^1 and 1.5×10^1 50% egg infective doses（EID_（50）） m L^–1, respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.展开更多
文摘目的为了提高微量样本中miRNA的检测通量和检测效率,本文建立了一种能够同时准确定量两种miRNA的双重实时荧光定量PCR(real time fluorogenic quantitative PCR)检测体系,并通过实际样本检测验证其应用于体液鉴别的效果。方法设计适用于miRNA双重检验的相关引物及探针并优化实验体系组分,建立基于TaqMan技术的miRNA双重实时荧光定量PCR检测体系并验证其特异性、灵敏度和可重复性;使用此检测体系对58份不同体液样本中的miR-451a与miR-21-5p进行检测,并借助miR-451a与miR-21-5p的比值鉴定法评估该体系的体液鉴别能力;使用该检测体系样本数据确定的最佳截断值对模拟案件样本进行鉴别。结果优化的检测体系能够实现对血液与非血液、月经血与外周血的100%区分,同时可以实现对模拟案件样本的准确鉴别。结论该双重实时荧光定量PCR检测体系将时间和材料成本均缩短至原来的一半,为后续建立更多重的实时荧光定量PCR检测体系并应用于体液鉴别打下了基础。
文摘为建立一种能快速对羊泰勒虫(ovine and caprine theileria)和嗜吞噬细胞无浆体(A.phagocytoph-ilum)同时进行检测的双重PCR方法。根据GenBank已报道的羊泰勒虫MPSP基因和羊嗜吞噬细胞无浆体16S rRNA基因设计合成了2对特异性引物,通过条件优化,建立了双重PCR检测方法。双重PCR可特异扩增出羊泰勒虫和嗜吞噬细胞无浆体目的条带,片段大小分别为875bp和394bp。该方法具有较好的特异性,对羊泰勒虫和嗜吞噬细胞无浆体的最低检出浓度为16fg/μL和1fg/μL。对采集的60份血液样本进行双重PCR检测,羊泰勒虫阳性率为46.67%(28/60),嗜吞噬细胞无浆体阳性率为13.33%(8/60),混合感染率为10%(6/60)。结果表明,双重PCR方法可用于羊泰勒虫和嗜吞噬细胞无浆体的快速诊断和流行病学调查。
基金supported by National Natural Science Foundation of China(No. 30771854)China-U.S.Collaborative Program on Emerging and Re-emerging Infectious Diseases(No. 1U2GGH000018-01)
文摘Objective:To identify members of genera of rickettsia and O.tsutsugammhi simultaneously.Methods:Rapid and duplex and nested PCR methods have been established by designing primers based on the conserved regions of heat shock protein GroEL gene.345 mouse viscera samples including liver,spleen and kidney,96 Xenopsylla cheopis and 32 chiggers collected from Hongta areas of Yuxi city,Yunnan province were tested by the new PCR methods.Results:The result of the study showed that the new PCR methods could identify most members of genera -Rickettsia and Orientia simultaneously with 100%specificity and its sensitivity could test one copy per microliter.The results of detection prevalence of rickettsioses in mouse,flea and mites DNA samples showed that the total rickettsia infection rate in mouse was 34.78%(120/345).The total infection rates in R.typhi,O.t Karp and R.sibirica of mouse samples were 28.12%(97/345),19.71%(68/345) and O. 29%(1/345) respectively.Co-infection rates in R.typhi and 0.t Karp of mouse samples were 13.33%(46/ 345).O.t Karp type has been the main epidemic strain in these areas.Conclusion:We concluded that this PCR method could be used to detect multi-genera rickettsia simultaneously.Molecular evidences provided in this and previous studies strongly support that Hongta areas of Yuxi city are a natural focus for typhus and scrub typhus with the common occurrence of their confection.
基金supported by Molecular Biology Research Center, Baqiyatallah University of Medical Sciences,with grant number BMSU/MBRC-90-001
文摘Objective:To design a duplex PCR for rapid and simultaneous detection of Brucella species, in human blood samples.Methods:Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis.Following DNA extraction,PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals.Results:Of the 52 peripheral bloods samples tested, 25 sample(48%) showed positive reactions in PCR.Twelve samples were positive for Brucella abortus(B.abortus)(23%,13 for Brucella melUensis(B.melUensis)(25%) and 0 for Brucella ovis (6.ovis)(Ow.Conclusions:This work de=monstrates dial in case where specific primers were utilized,duplex PCR has proved to be a simple,fast,and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples.
基金supported by the National High-Tech R&D Program of China(2012AA101303)
文摘H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction（RT-PCR） assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR（d RT-PCR） was established. Two primer sets target the hemagglutinin（HA） gene of H9 AIV and the nucleocapsid（N） gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10^1, 1.5×10^1 and 1.5×10^1 50% egg infective doses（EID_（50）） m L^–1, respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.
