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
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
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
A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) was developed for the detection of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV) and porcine ...A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) was developed for the detection of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV) and porcine group A rotavirus (GAR). Three pairs of primers were designed to target the M gene, N gene, and VP7 gene of PEDV, TGEV, GAR, respectively, and the multiplex RT-PCR was developed and optimized. The results of the multiplex RT-PCR and routine single RT-PCRs were compared using samples collected in the field. In laboratory testing, the detection limit of the multiplex RT-PCR is ～35 pg RNA of combined TGEV-PEDV-GAR vaccine. In the field trial, 75 fecal specimens collected from pigs with diarrhea, in the central area of China, were simultaneously tested by the multiplex RT-PCR and by routine single RT-PCRs to evaluate the relative sensitivity and specificity of the multiplex RT-PCR. The results indicate that this new assay is equal in quality to the routine RT-PCR assays (sensitivities were 92%, 100%, 100% for PEDV, TGEV, GAR, respectively; specificity was 100% for all three viruses). The multiplex RT-PCR, with high sensitivity and specificity, provides a new and alternative tool for the detection of PEDV, TGEV and GAR.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.
文摘A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) was developed for the detection of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV) and porcine group A rotavirus (GAR). Three pairs of primers were designed to target the M gene, N gene, and VP7 gene of PEDV, TGEV, GAR, respectively, and the multiplex RT-PCR was developed and optimized. The results of the multiplex RT-PCR and routine single RT-PCRs were compared using samples collected in the field. In laboratory testing, the detection limit of the multiplex RT-PCR is ～35 pg RNA of combined TGEV-PEDV-GAR vaccine. In the field trial, 75 fecal specimens collected from pigs with diarrhea, in the central area of China, were simultaneously tested by the multiplex RT-PCR and by routine single RT-PCRs to evaluate the relative sensitivity and specificity of the multiplex RT-PCR. The results indicate that this new assay is equal in quality to the routine RT-PCR assays (sensitivities were 92%, 100%, 100% for PEDV, TGEV, GAR, respectively; specificity was 100% for all three viruses). The multiplex RT-PCR, with high sensitivity and specificity, provides a new and alternative tool for the detection of PEDV, TGEV and GAR.
