TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybean...TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x＋35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x＋35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.展开更多
A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gen...A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gene expression at low temperature, and ultimately for assigning a role for the gene in the low temperature autolysis of V. volvacea.展开更多
AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed. To better understand cellular functions, the isolation of single cells and subsequen...AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed. To better understand cellular functions, the isolation of single cells and subsequent quantification of the expressed genes is essential. METHODS: Normal liver tissue was obtained from operation, snap-frozen in liquid nitrogen and sectioned in crystat. Individual hepatocytes were microdissected. RNA was extracted, then reverse transcribed and amplified using real-time quantitative polymerase chain reaction (PCR). RESULTS: Single hepatocytes were dissected by laser beam and catapulted to the microcentrifuge cap which was put above the slide. In this way, cells were collected, RNA was extracted, reverse transcribed to cDNA and used for analysis of RNA expression by real-time quantitative PCR. The amplification results showed that quantitation of the RNA inside the cell was compatible with the number of cells. CONCLUSION: The expression of RNA in single cells can be quantitated successfully by using laser microdissection and real-time PCR. These techniques provide an opportunity to monitor in vivo gene expression levels in single hepatocytes.展开更多
目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-...目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。展开更多
应用SYBR Green I染料能选择性结合双链DNA的特点,可检测到沙门氏菌fimI基因特异性靶序列扩增所产生的荧光信号,通过熔解曲线可知其熔点值约为85.6℃,而对其他非沙门氏菌则检测不到荧光信号。建立了一种肉品中的沙门氏菌Real-time PCR...应用SYBR Green I染料能选择性结合双链DNA的特点,可检测到沙门氏菌fimI基因特异性靶序列扩增所产生的荧光信号,通过熔解曲线可知其熔点值约为85.6℃,而对其他非沙门氏菌则检测不到荧光信号。建立了一种肉品中的沙门氏菌Real-time PCR检测方法,用该方法检测市售牛肉、香肠中的沙门氏菌,其检测灵敏度分别为13,12 cfu/25 g,从样品的处理到得出检验结果可以在10 h内完成。该检测方法具有简便、快速、特异性强、敏感度高等特点。展开更多
基金Supported by the Program of Technology Bureau of Harbin (2010RFQXN101)the Subproject of Transgenic Significant Specific Project (20112X08004-002-002-004)
文摘TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x＋35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x＋35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.
基金Sponsored by the Shanghai Academy of Agricultural Science and Shanghai Science Committee (No .003912076)
文摘A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gene expression at low temperature, and ultimately for assigning a role for the gene in the low temperature autolysis of V. volvacea.
文摘AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed. To better understand cellular functions, the isolation of single cells and subsequent quantification of the expressed genes is essential. METHODS: Normal liver tissue was obtained from operation, snap-frozen in liquid nitrogen and sectioned in crystat. Individual hepatocytes were microdissected. RNA was extracted, then reverse transcribed and amplified using real-time quantitative polymerase chain reaction (PCR). RESULTS: Single hepatocytes were dissected by laser beam and catapulted to the microcentrifuge cap which was put above the slide. In this way, cells were collected, RNA was extracted, reverse transcribed to cDNA and used for analysis of RNA expression by real-time quantitative PCR. The amplification results showed that quantitation of the RNA inside the cell was compatible with the number of cells. CONCLUSION: The expression of RNA in single cells can be quantitated successfully by using laser microdissection and real-time PCR. These techniques provide an opportunity to monitor in vivo gene expression levels in single hepatocytes.
文摘目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。
