Classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus （PRRSV） have caused immense economic loss in the pig industry and are considered to be the two most important infectious d...Classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus （PRRSV） have caused immense economic loss in the pig industry and are considered to be the two most important infectious diseases of pigs in the world A multiplex reverse transcription polymerase chain reaction （multiplex RT-PCR） was developed for CSFV and PRRSV co-infections or infections, respectively. A set of two pairs of primer was designed based on the sequence of nonstructural protein NS54B of CSFV and ORF7 gene of PRRSV. The diagnostic accuracy of multiplex RT-PCR assay was evaluated by using 56 field clinical samples by multiplex RT-PCR, single RT-PCR and sequence analysis; and the specificity of multiplex PCR was verified by using constructed plasmids containing the specific viral target fragments of PRRSV and CSFV, respectively. The results indicated that this assay could reliably differentiate PRRSV and CSFV in co-infection samples. The multiplex RT-PCR developed in this study might provide a new avenue to the rapid the detection of CSFV and PRRSV in one reaction.展开更多
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
As the combining form of the orthogonal frequency-division multiplexing (OFDM) technique and the vertical Bell Labs layered space-time (V-BLAST) architecture, the V-BLAST OFDM system can better meet the demand of next...As the combining form of the orthogonal frequency-division multiplexing (OFDM) technique and the vertical Bell Labs layered space-time (V-BLAST) architecture, the V-BLAST OFDM system can better meet the demand of next-generation (NextG) broadband mobile wireless multimedia communications. The symbols detection problem of the V-BLAST OFDM system is investigated under the frequency-selective fading environment. The joint space-frequency demultiplexing operation is proposed in the V-BLAST OFDM system. Successively, one novel half-rate rotational invariance joint space-frequency coding scheme for the V-BLAST OFDM system is proposed. By elegantly exploiting the above rotational invariance property, we derive one direct symbols detection scheme without knowing channels state information (CSI) for the frequency-selective V-BLAST OFDM system. Extensive simulation results demonstrate the validity of the novel half-rate rotational invariance joint space-frequency coding scheme and the performance of the direct symbols detection scheme.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
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
为建立并比较牛传染性鼻气管炎病毒(IBRV)的检测方法,本研究基于IBRV gB基因和gE基因的保守序列,分别设计用于检测IBRV的TB Green I荧光定量PCR(qPCR)方法和纳米(Nano)PCR方法的特异性引物,利用本实验构建的重组质粒标准品pMD19-T-gB和p...为建立并比较牛传染性鼻气管炎病毒(IBRV)的检测方法,本研究基于IBRV gB基因和gE基因的保守序列,分别设计用于检测IBRV的TB Green I荧光定量PCR(qPCR)方法和纳米(Nano)PCR方法的特异性引物,利用本实验构建的重组质粒标准品pMD19-T-gB和pMD19-T-gE为模板,采用方阵法优化各反应条件,初步建立了检测IBRV的TB Green I qPCR方法和Nano PCR方法,并建立荧光定量PCR标准曲线。以牛病毒性腹泻病毒(BVDV)、牛副流感病毒3型(BPIV3)、牛肠道病毒(BEV)和牛流行热病毒(BEFV)等RNA反转录所得cDNA及IBRV基因组DNA为模板,采用本研究建立的TB Green I qPCR方法和Nano PCR方法检测,评估所建方法的特异性;分别以10倍倍比稀释(108~101)的pMD19-T-gB和pMD19-T-gE质粒标准品作为模板,利用本研究建立的两种方法与普通PCR方法检测,比较所建方法的敏感性;以3个不同浓度的重组质粒标准品pMD19-T-gB作为模板,利用TB Green I qPCR方法分别于同一时间和不同时间进行批内和批间重复性试验,评估该方法的重复性。标准曲线结果显示,质粒标准品pMD19-T-gB在5.54×10^(8)拷贝/μL~5.54×10^(1)拷贝/μL范围内与其Ct值之间的线性关系良好,相关系数R2=0.999,溶解曲线为单一峰。特异性试验结果显示,所建立的两种方法仅能检出IBRV,其他病毒均为阴性结果,特异性强。敏感性试验结果显示,所建立的TB Green I qPCR方法对重组质粒标准品pMD19-T-gB的检测限为55.4拷贝/μL;Nano PCR方法对重组质粒标准品pMD19-T-gE的检测限为4.28×10^(3)拷贝/μL,所建立两种方法的敏感性均较高。重复性试验结果显示,该qPCR方法批内、批间重复性试验的变异系数在0.16%~0.67%,重复性较好。采用本实验建立的两种方法和普通PCR方法对延边地区采集的50份疑似IBRV感染的样品进行检测,结果显示,普通PCR对样品的阳性检测率为84%(42/50),Nano PCR对样品的阳性检测率为90%(45/50),qPCR对样品的阳性检测率为92%(46/50),3种检测方法的总符合率均高于85%。本研究建立的qPCR方法对样品的阳性检出率最高、特异性更强,为临床样品的快速检测及相应疾病早期诊断的首选;Nano PCR次之。本实验首次同时建立并比较了用于IBRV检测的TB Green I qPCR方法与Nano PCR方法,且首次对延边地区的IBRV流行病学作了初步调查,为牛养殖业和疫病防控工作提供技术支撑和参考依据。展开更多
文摘Classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus （PRRSV） have caused immense economic loss in the pig industry and are considered to be the two most important infectious diseases of pigs in the world A multiplex reverse transcription polymerase chain reaction （multiplex RT-PCR） was developed for CSFV and PRRSV co-infections or infections, respectively. A set of two pairs of primer was designed based on the sequence of nonstructural protein NS54B of CSFV and ORF7 gene of PRRSV. The diagnostic accuracy of multiplex RT-PCR assay was evaluated by using 56 field clinical samples by multiplex RT-PCR, single RT-PCR and sequence analysis; and the specificity of multiplex PCR was verified by using constructed plasmids containing the specific viral target fragments of PRRSV and CSFV, respectively. The results indicated that this assay could reliably differentiate PRRSV and CSFV in co-infection samples. The multiplex RT-PCR developed in this study might provide a new avenue to the rapid the detection of CSFV and PRRSV in one reaction.
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As the combining form of the orthogonal frequency-division multiplexing (OFDM) technique and the vertical Bell Labs layered space-time (V-BLAST) architecture, the V-BLAST OFDM system can better meet the demand of next-generation (NextG) broadband mobile wireless multimedia communications. The symbols detection problem of the V-BLAST OFDM system is investigated under the frequency-selective fading environment. The joint space-frequency demultiplexing operation is proposed in the V-BLAST OFDM system. Successively, one novel half-rate rotational invariance joint space-frequency coding scheme for the V-BLAST OFDM system is proposed. By elegantly exploiting the above rotational invariance property, we derive one direct symbols detection scheme without knowing channels state information (CSI) for the frequency-selective V-BLAST OFDM system. Extensive simulation results demonstrate the validity of the novel half-rate rotational invariance joint space-frequency coding scheme and the performance of the direct symbols detection scheme.
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。
文摘为建立并比较牛传染性鼻气管炎病毒(IBRV)的检测方法,本研究基于IBRV gB基因和gE基因的保守序列,分别设计用于检测IBRV的TB Green I荧光定量PCR(qPCR)方法和纳米(Nano)PCR方法的特异性引物,利用本实验构建的重组质粒标准品pMD19-T-gB和pMD19-T-gE为模板,采用方阵法优化各反应条件,初步建立了检测IBRV的TB Green I qPCR方法和Nano PCR方法,并建立荧光定量PCR标准曲线。以牛病毒性腹泻病毒(BVDV)、牛副流感病毒3型(BPIV3)、牛肠道病毒(BEV)和牛流行热病毒(BEFV)等RNA反转录所得cDNA及IBRV基因组DNA为模板,采用本研究建立的TB Green I qPCR方法和Nano PCR方法检测,评估所建方法的特异性;分别以10倍倍比稀释(108~101)的pMD19-T-gB和pMD19-T-gE质粒标准品作为模板,利用本研究建立的两种方法与普通PCR方法检测,比较所建方法的敏感性;以3个不同浓度的重组质粒标准品pMD19-T-gB作为模板,利用TB Green I qPCR方法分别于同一时间和不同时间进行批内和批间重复性试验,评估该方法的重复性。标准曲线结果显示,质粒标准品pMD19-T-gB在5.54×10^(8)拷贝/μL~5.54×10^(1)拷贝/μL范围内与其Ct值之间的线性关系良好,相关系数R2=0.999,溶解曲线为单一峰。特异性试验结果显示,所建立的两种方法仅能检出IBRV,其他病毒均为阴性结果,特异性强。敏感性试验结果显示,所建立的TB Green I qPCR方法对重组质粒标准品pMD19-T-gB的检测限为55.4拷贝/μL;Nano PCR方法对重组质粒标准品pMD19-T-gE的检测限为4.28×10^(3)拷贝/μL,所建立两种方法的敏感性均较高。重复性试验结果显示,该qPCR方法批内、批间重复性试验的变异系数在0.16%~0.67%,重复性较好。采用本实验建立的两种方法和普通PCR方法对延边地区采集的50份疑似IBRV感染的样品进行检测,结果显示,普通PCR对样品的阳性检测率为84%(42/50),Nano PCR对样品的阳性检测率为90%(45/50),qPCR对样品的阳性检测率为92%(46/50),3种检测方法的总符合率均高于85%。本研究建立的qPCR方法对样品的阳性检出率最高、特异性更强,为临床样品的快速检测及相应疾病早期诊断的首选;Nano PCR次之。本实验首次同时建立并比较了用于IBRV检测的TB Green I qPCR方法与Nano PCR方法,且首次对延边地区的IBRV流行病学作了初步调查,为牛养殖业和疫病防控工作提供技术支撑和参考依据。
