Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. ...Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components (enzyme, MgCl2, DNA template and dNTPs) in PCR system were used in the research with the concentration of the other component remained the same; the orthogonal design L9 (34) was applied in the optimization of four sets of primers (STM0014, Pat, SSI, and UGP) in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL : 2.5 μL 25 mmol.L-1 MgCl2, 0.6 μL 10 mmol·L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol. L1 primers was 2 : 1 : 2 : 3, and the annealing temperature was 54.7℃. The optimized reaction system could be repeated stably; and the stable and reliable amplification results were able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..展开更多
十八微卫星教材对以前为 Populus tremuloides Michx 在橡树理兹公民实验室发展了。并且 Populus trichocarpa Torr。与格雷在幼发拉底河白杨为扩大被屏蔽, Populus euphratica Oliv。13 loci 被发现表示从二到 17 等位基因的多型性。...十八微卫星教材对以前为 Populus tremuloides Michx 在橡树理兹公民实验室发展了。并且 Populus trichocarpa Torr。与格雷在幼发拉底河白杨为扩大被屏蔽, Populus euphratica Oliv。13 loci 被发现表示从二到 17 等位基因的多型性。八很可变的 loci 被选择建立并且优化二复合聚合酶链反应(PCR ) 试金。总共包含 436 棵树的三张人口被用来描绘选择 loci 并且为出身分析查明他们的适用性, genotyping 学习。通过对 genotyped 树的性的同种细胞的身份的 cross-checking,我们为合并遗传型是不到 0.045 估计了最大的错误率。展开更多
Objective:To evaluate the use of Real-Time PCR system based on specific amplification of matrix protein gene fragment for influenza A virus RNA detection in cloacal swabs from wild birds.Methods:Sensitivity,specificit...Objective:To evaluate the use of Real-Time PCR system based on specific amplification of matrix protein gene fragment for influenza A virus RNA detection in cloacal swabs from wild birds.Methods:Sensitivity,specificity and reproducibility of analysis results were identified. Study of cloacal swabs from wild birds for influenza A virus presence was performed.Results: Reproducibility of low concentrations of virus detection in samples by Real-Time PCR was significantly higher than that of detection based on cytopathic effect of viruses grown on MDCK cell culture.Conclusions:Real-Time PCR system for influenza A virus RNA detection is developed and applied for virus surveillance study.展开更多
Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (...Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (FHCM).A microarray was fabricated to screen mutations in exons 3,5,7,and 8 in cTnI gene.Primers were designed for the PCR (polymerase chain reaction) to amplify the target DNA fragments from fresh blood samples.In order to simplify the PCR process,multiplex PCR technology was investigated in detail.The concentration of Mg^(2+) played an important role in multiplex PCR process,a properly low concentration of Mg^(2+) submitted a better speciality of PCR products.The speciality was also favored when the annealing temperature was reasonably enhanced and 64℃is the optimal annealing temperature for the multiplex PCR systems.When applying the fabricated gene-chip to detect the target fragments from PCR mixture,the signal intensity sequence is in accordance with that from theoretic estimate.展开更多
Late blight caused by the oomycete Phytophthora infestans Montagne de Bary is a devastating disease on potato production. A total of six parameters affecting the PCR system in P. infestans were investigated based on t...Late blight caused by the oomycete Phytophthora infestans Montagne de Bary is a devastating disease on potato production. A total of six parameters affecting the PCR system in P. infestans were investigated based on the template DNA of the isolate HH06-23 and EST-SSR primer pair Pi08N. The results showed that the optimal annealing temperature was 63 ℃, and the optimum PCR system of EST-SSR was 25 ng template DNA, 0.5 mmol·L^-1 dNTPs, 2 μL 10×Buffer (Mg^2+ free), 1.75 mmol·L^-1 MgCl2, 15 pmol primer, and 1.2 U Taq DNA polymerase in total 20 μL reaction system. The PCR program was initial denaturation at 94℃ for 2 rain, followed by 35 cycles of 94℃ for 30 s, 63℃ for 30 s, and 72℃ for 30 s, then a final extension step was 72℃ for 7 min, and held at 4℃. In addition, using the optimal PCR system, a total of 20 isolates of P. infestans were used for testing the stability and polymorphism of the PCR amplification. The clarity and abundant polymorphism indicated that this system was stable and suitable for researching the genetic diversity ofP. infestans population.展开更多
This study was conducted to develop a method for accurate quantification of Mycoplasma hyopneumoniae during vaccine production or experimental research. Primer and probe concentration that gave the highest ΔRn and th...This study was conducted to develop a method for accurate quantification of Mycoplasma hyopneumoniae during vaccine production or experimental research. Primer and probe concentration that gave the highest ΔRn and the lowest Ct were selected to establish the real-time PCR system for the detection of M. hyopneumoniae. Template DNA of M. hyopneumoniae was extracted by boiling under different conditions and detected by real-time PCR to determine the optimal conditions for DNA extraction. Thereafter, intra-and inter-batch reproducibility tests were carried out using a standard plasmid to evaluate the stability of the PCR system. Subsequently, the effect of medium composition on the quantitative detection was evaluated. Finally, the correlation between real-time PCR and CCU method was explored. The optimal primer and probe concentration for real-time PCR were 0.4 and 0.2 μmol/L, respectively. The intra-and inter-batch coefficients of variation(CV) in Ct value of 10~4-10~9 copies/μl standard plasmid were <5%, indicating good reproducibility of the real-time PCR system. Following incubation in a boiling water bath for 10 min, M. hyopneumoniae samples can be used directly as a template in subsequent real-time PCR assays,and good intra-batch and inter-batch reproducibility was observed. The working concentration of KM2 medium should be less than the 1/10 of the concentration of the stock solution to minimize its influence on the quantitative detection. Spearman's correlation analysis revealed that the log of CCU and the log of DNA copy number had a significant positive relationship(r=0.797,P=0.000). Thus, the two methods can be used in combination in the quantitative detection of M. hyopneumoniae. In summary, a rapid, stable and accurate quantitative PCR system for detecting M. hyopneumoniae culture was established in this study, which provides a technical means for accurate quantification of M. hyopneumoniae in vaccine production and laboratory tests.展开更多
[Objective] This study aimed to establish a quantitative real-time PCR(qRT-PCR) system for detecting the expression of rice beta-glucosidase gene Os1bglu4. [Method] The PCR was conducted with SYBR Green I method, usin...[Objective] This study aimed to establish a quantitative real-time PCR(qRT-PCR) system for detecting the expression of rice beta-glucosidase gene Os1bglu4. [Method] The PCR was conducted with SYBR Green I method, using the primers of reference gene actin or ubiquitin. [Result] Actin was more suitable to be the reference gene than ubiquitin. More accurate results were obtained when the 100 ng cDNA template was added at a large volume and a lower concentration.The primer concentration in the range from 0.2 to 0.8 μmol/L we set had no significant influence on the results, so, 0.4 μmol/L was selected as the optimal primer concentration in this study. The amplification efficiency was greatly reduced when the annealing temperature was set at 64 ℃, therefore, annealing temperature was set at 60 ℃. Compared with the reaction system of 25 μl, the fluorescence intensity was significantly lower but the CT value did not change greatly in 10 μl system.So, the 10 μl reaction system was selected, which significantly reduces the research costs for the detection of a large amount of samples in future study.展开更多
With Portulaca oleracea L. as an experimental material, its total DNA was extracted by the improved CTAB method, the ISSR-PCR primers were screened, and the ISSR-PCR reaction system and reaction conditions for P. oler...With Portulaca oleracea L. as an experimental material, its total DNA was extracted by the improved CTAB method, the ISSR-PCR primers were screened, and the ISSR-PCR reaction system and reaction conditions for P. oleracea were Optimized. The results showed that there were 8 primers suitable for ISSR-PCR of P. oleracea. The optimal reaction system had a volume of 25 μl, including 2 x Taq Platinum PCR Master Mix 12.5 μl, primer 2 μl, ddH20 9.5 μl, and DNA template 1μl. The optimized ISSR-PCR of P. oleracea was started with pre-denaturation at 94 ℃ for 360 s, followed by 30 cycles of denaturation at 94 ℃ for 60 s, annealing at 54 ℃ for 60 s and extension at 72 ℃ for 90 s, and completed by extension at 72 ℃ for 300 s.展开更多
为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法。该方法的扩增效率(E)为90.0%,相关系数(R~2)=0.99,标准曲线方程为y=-3.607x+38....为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法。该方法的扩增效率(E)为90.0%,相关系数(R~2)=0.99,标准曲线方程为y=-3.607x+38.77;除WPV出现S形扩增曲线外,新城疫病毒(NDV)、H9亚型禽流感病毒(H9 AIV)、鸭坦布苏病毒(DTMUV)、鸭肝炎病毒(DHAV)、鸭肠炎病毒(DEV)、鸭呼肠孤病毒(DRV)样品均未出现S形阳性扩增曲线;批内变异系数(CV)为0.15%~0.23%,批间变异系数为0.09%~0.28%。结果表明,SYBR Green Ⅰ荧光定量PCR检测方法重复性好、灵敏度高和特异性强。临床样品检测结果表明,SYBR Green Ⅰ荧光定量PCR与普通PCR的符合率达98.4%,灵敏度是普通PCR的1 000倍。SYBR Green Ⅰ荧光定量PCR检测方法不仅能定性检测WPV,还可以进行定量检测,可用于种鸭场、种鹅场的WPV净化检测,也可用于WPV临床大量样品的快速检测。展开更多
基金Supported by the International Cooperation Project of Heilongjiang Science and Technology Department (WC05B08)
文摘Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components (enzyme, MgCl2, DNA template and dNTPs) in PCR system were used in the research with the concentration of the other component remained the same; the orthogonal design L9 (34) was applied in the optimization of four sets of primers (STM0014, Pat, SSI, and UGP) in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL : 2.5 μL 25 mmol.L-1 MgCl2, 0.6 μL 10 mmol·L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol. L1 primers was 2 : 1 : 2 : 3, and the annealing temperature was 54.7℃. The optimized reaction system could be repeated stably; and the stable and reliable amplification results were able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..
文摘十八微卫星教材对以前为 Populus tremuloides Michx 在橡树理兹公民实验室发展了。并且 Populus trichocarpa Torr。与格雷在幼发拉底河白杨为扩大被屏蔽, Populus euphratica Oliv。13 loci 被发现表示从二到 17 等位基因的多型性。八很可变的 loci 被选择建立并且优化二复合聚合酶链反应(PCR ) 试金。总共包含 436 棵树的三张人口被用来描绘选择 loci 并且为出身分析查明他们的适用性, genotyping 学习。通过对 genotyped 树的性的同种细胞的身份的 cross-checking,我们为合并遗传型是不到 0.045 估计了最大的错误率。
文摘Objective:To evaluate the use of Real-Time PCR system based on specific amplification of matrix protein gene fragment for influenza A virus RNA detection in cloacal swabs from wild birds.Methods:Sensitivity,specificity and reproducibility of analysis results were identified. Study of cloacal swabs from wild birds for influenza A virus presence was performed.Results: Reproducibility of low concentrations of virus detection in samples by Real-Time PCR was significantly higher than that of detection based on cytopathic effect of viruses grown on MDCK cell culture.Conclusions:Real-Time PCR system for influenza A virus RNA detection is developed and applied for virus surveillance study.
文摘Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (FHCM).A microarray was fabricated to screen mutations in exons 3,5,7,and 8 in cTnI gene.Primers were designed for the PCR (polymerase chain reaction) to amplify the target DNA fragments from fresh blood samples.In order to simplify the PCR process,multiplex PCR technology was investigated in detail.The concentration of Mg^(2+) played an important role in multiplex PCR process,a properly low concentration of Mg^(2+) submitted a better speciality of PCR products.The speciality was also favored when the annealing temperature was reasonably enhanced and 64℃is the optimal annealing temperature for the multiplex PCR systems.When applying the fabricated gene-chip to detect the target fragments from PCR mixture,the signal intensity sequence is in accordance with that from theoretic estimate.
基金Supported by Natural Science Foundation of Heilongjiang Province of China (C200622)
文摘Late blight caused by the oomycete Phytophthora infestans Montagne de Bary is a devastating disease on potato production. A total of six parameters affecting the PCR system in P. infestans were investigated based on the template DNA of the isolate HH06-23 and EST-SSR primer pair Pi08N. The results showed that the optimal annealing temperature was 63 ℃, and the optimum PCR system of EST-SSR was 25 ng template DNA, 0.5 mmol·L^-1 dNTPs, 2 μL 10×Buffer (Mg^2+ free), 1.75 mmol·L^-1 MgCl2, 15 pmol primer, and 1.2 U Taq DNA polymerase in total 20 μL reaction system. The PCR program was initial denaturation at 94℃ for 2 rain, followed by 35 cycles of 94℃ for 30 s, 63℃ for 30 s, and 72℃ for 30 s, then a final extension step was 72℃ for 7 min, and held at 4℃. In addition, using the optimal PCR system, a total of 20 isolates of P. infestans were used for testing the stability and polymorphism of the PCR amplification. The clarity and abundant polymorphism indicated that this system was stable and suitable for researching the genetic diversity ofP. infestans population.
基金Supported by National Key Research and Development Project of China(2017YFD0501604)National Natural Science Foundation of China(31400164)
文摘This study was conducted to develop a method for accurate quantification of Mycoplasma hyopneumoniae during vaccine production or experimental research. Primer and probe concentration that gave the highest ΔRn and the lowest Ct were selected to establish the real-time PCR system for the detection of M. hyopneumoniae. Template DNA of M. hyopneumoniae was extracted by boiling under different conditions and detected by real-time PCR to determine the optimal conditions for DNA extraction. Thereafter, intra-and inter-batch reproducibility tests were carried out using a standard plasmid to evaluate the stability of the PCR system. Subsequently, the effect of medium composition on the quantitative detection was evaluated. Finally, the correlation between real-time PCR and CCU method was explored. The optimal primer and probe concentration for real-time PCR were 0.4 and 0.2 μmol/L, respectively. The intra-and inter-batch coefficients of variation(CV) in Ct value of 10~4-10~9 copies/μl standard plasmid were <5%, indicating good reproducibility of the real-time PCR system. Following incubation in a boiling water bath for 10 min, M. hyopneumoniae samples can be used directly as a template in subsequent real-time PCR assays,and good intra-batch and inter-batch reproducibility was observed. The working concentration of KM2 medium should be less than the 1/10 of the concentration of the stock solution to minimize its influence on the quantitative detection. Spearman's correlation analysis revealed that the log of CCU and the log of DNA copy number had a significant positive relationship(r=0.797,P=0.000). Thus, the two methods can be used in combination in the quantitative detection of M. hyopneumoniae. In summary, a rapid, stable and accurate quantitative PCR system for detecting M. hyopneumoniae culture was established in this study, which provides a technical means for accurate quantification of M. hyopneumoniae in vaccine production and laboratory tests.
基金Supported by Guizhou International Cooperation Project on Science and Technology[(2013)7040]the 20th Project of the Joint Committee on Scientific and Technical Cooperation between the Government of the Kingdom of Thailand and the Government of the People’s Republic of China (20-606J)the Fund from Suranaree University of Technology,Thailand (SUT3-304-54-12-29)
文摘[Objective] This study aimed to establish a quantitative real-time PCR(qRT-PCR) system for detecting the expression of rice beta-glucosidase gene Os1bglu4. [Method] The PCR was conducted with SYBR Green I method, using the primers of reference gene actin or ubiquitin. [Result] Actin was more suitable to be the reference gene than ubiquitin. More accurate results were obtained when the 100 ng cDNA template was added at a large volume and a lower concentration.The primer concentration in the range from 0.2 to 0.8 μmol/L we set had no significant influence on the results, so, 0.4 μmol/L was selected as the optimal primer concentration in this study. The amplification efficiency was greatly reduced when the annealing temperature was set at 64 ℃, therefore, annealing temperature was set at 60 ℃. Compared with the reaction system of 25 μl, the fluorescence intensity was significantly lower but the CT value did not change greatly in 10 μl system.So, the 10 μl reaction system was selected, which significantly reduces the research costs for the detection of a large amount of samples in future study.
文摘With Portulaca oleracea L. as an experimental material, its total DNA was extracted by the improved CTAB method, the ISSR-PCR primers were screened, and the ISSR-PCR reaction system and reaction conditions for P. oleracea were Optimized. The results showed that there were 8 primers suitable for ISSR-PCR of P. oleracea. The optimal reaction system had a volume of 25 μl, including 2 x Taq Platinum PCR Master Mix 12.5 μl, primer 2 μl, ddH20 9.5 μl, and DNA template 1μl. The optimized ISSR-PCR of P. oleracea was started with pre-denaturation at 94 ℃ for 360 s, followed by 30 cycles of denaturation at 94 ℃ for 60 s, annealing at 54 ℃ for 60 s and extension at 72 ℃ for 90 s, and completed by extension at 72 ℃ for 300 s.
文摘为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法。该方法的扩增效率(E)为90.0%,相关系数(R~2)=0.99,标准曲线方程为y=-3.607x+38.77;除WPV出现S形扩增曲线外,新城疫病毒(NDV)、H9亚型禽流感病毒(H9 AIV)、鸭坦布苏病毒(DTMUV)、鸭肝炎病毒(DHAV)、鸭肠炎病毒(DEV)、鸭呼肠孤病毒(DRV)样品均未出现S形阳性扩增曲线;批内变异系数(CV)为0.15%~0.23%,批间变异系数为0.09%~0.28%。结果表明,SYBR Green Ⅰ荧光定量PCR检测方法重复性好、灵敏度高和特异性强。临床样品检测结果表明,SYBR Green Ⅰ荧光定量PCR与普通PCR的符合率达98.4%,灵敏度是普通PCR的1 000倍。SYBR Green Ⅰ荧光定量PCR检测方法不仅能定性检测WPV,还可以进行定量检测,可用于种鸭场、种鹅场的WPV净化检测,也可用于WPV临床大量样品的快速检测。