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  fluorescent signal
The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0.
      
Methods enabling designing of probes with a minimum initial level of the fluorescent signal are examined and analyzed.
      
At later time-points the fluorescent signal appeared granular; at these times the injected material was largely degraded.
      
Different marker selection thresholds for average fluorescent signal intensity and marker frequency were used to create eight extra restricted data subsets.
      
The specifity of the fluorescent signal and the Chromatographic conditions allow to work without any special preparation of the sample.
      
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A real time fluorescent PCR(RTF PCR) method was established to detect and identify phytoplasmas. One universal and three group specific TaqMan probes were designed based on the conserved region of the 16S rDNA of phytoplasmas,and they were used to detect 9 strains of phytoplasmas and 5 species of bacteria and three samples by RTF PCR. The results showed that the universal probe could detect all the phytoplasmas used, while no signal was detected for bacteria. For three group specific probes, each could detect...

A real time fluorescent PCR(RTF PCR) method was established to detect and identify phytoplasmas. One universal and three group specific TaqMan probes were designed based on the conserved region of the 16S rDNA of phytoplasmas,and they were used to detect 9 strains of phytoplasmas and 5 species of bacteria and three samples by RTF PCR. The results showed that the universal probe could detect all the phytoplasmas used, while no signal was detected for bacteria. For three group specific probes, each could detect its own group specifically. The method was 100 times more sensitive than normal PCR and more specific and much faster due to fluorescent probe used. It could even identify the phytoplasma species while the group specific probes used. Few contamination would occur because whole detection process was finished in the contained tubes. RTF PCR could be a new technique for detecting other prokaryotes, especially the fastidious bacteria.

本研究成功建立了植原体分类鉴定和检测的 Taq Man探针实时荧光 PCR方法 ,该方法根据植原体 16S r DNA保守区设计了 1个 Taq Man广谱探针和 3个植原体组间点突变特异性探针 ,并对 9种植原体和 5种细菌以及 3个植物样本进行实时荧光 PCR。结果表明 ,用广谱探针可检测到所有植原体产生荧光信号 ,而细菌不产生荧光信号。当用植原体组间特异性探针检测时 ,仅能检测到该组植原体产生荧光信号 ,检测的敏感性比常规的 PCR-电泳检测高约 10 0倍、检测速度有较大提高。由于PCR产物是荧光探针检测 ,本方法特异性强 ,并可以用组特异探针直接确定植原体种类。实验采用完全闭管检测 ,降低了污染机会。本研究为其它原核生物、特别是不能培养菌、难培养菌的检测鉴定和分类提供了新方法

A novel and sensitive real time PCR was developed to detection \%Xanthomonas ory zae\% pv.\%oryzae\% and \%Xanthomonas oryzae\% pv.\%oryzicola\%, which cause the bacteria leaf blight (BLB) and leaf streak respectively, Universal and specific TaqMan probes, which were designed based on the sequence of Putative siderophor e receptor gene cds were used to detect 13 bacteria and one phytoplasmas, only in \%X.oryzae \%pv. \%oryzae\% and \%X.oryzae \%pv. \%oryzicola\%, fluorescen t signal can be collected with...

A novel and sensitive real time PCR was developed to detection \%Xanthomonas ory zae\% pv.\%oryzae\% and \%Xanthomonas oryzae\% pv.\%oryzicola\%, which cause the bacteria leaf blight (BLB) and leaf streak respectively, Universal and specific TaqMan probes, which were designed based on the sequence of Putative siderophor e receptor gene cds were used to detect 13 bacteria and one phytoplasmas, only in \%X.oryzae \%pv. \%oryzae\% and \%X.oryzae \%pv. \%oryzicola\%, fluorescen t signal can be collected with their specific probes respectively. The level of detection of the probe was 30.6fg plasmid, roughly equaling to one cell and 100 times sensit ive than PCR gel electrophoresis detection.\% X.oryzae \%pv. \%oryzae\% and \% X.oryzae \%pv \%oryzicola\% were detected from seed washes and DNA extracted fro m the seed washes of naturally infected seeds and in fected leaves as small as 10g naturally infected seeds or 0.3g leaf. This method is little time consumption (only 2h) and without contamination from PCR product .

成功建立了水稻白叶枯菌与水稻细菌性条斑病菌快速检测鉴定的实时荧光PCR方法。根据含铁细胞接受子基因设计两菌的通用引物PSRGF PSRGR(扩增一个 1 5 2bpDNA片段 )和特异性探针 (Baiprobe和Tiaoprobe) ,并对 1 3种细菌和 1种植原体进行实时荧光PCR。结果表明 ,两个特异性探针能分别特异性检测到目标病原菌产生荧光信号而其它参考菌不产生荧光信号。检测的绝对灵敏度是 3 0 .6fg μL质粒DNA和 1 0 3CFU mL的菌悬浮液 ,相当于 1个细菌细胞的基因 ,比常规PCR电泳检测高约 1 0 0倍 ,相对灵敏度为 1 0 5CFU mL。整个检测过程只需 2h ,完全闭管 ,降低了污染的机会 ,无需PCR后处理。用这两个特异性探针分别对自然感染白叶枯菌和条斑菌的叶片DNA提取液和种子浸泡液进行实时荧光PCR ,结果均可特异性检测到目标菌的存在并完全可将两种病原细菌区分开来 ,且只需 0 3g叶片和 1 0g种子

A very serious quarantine bacterium(on the A2 list of China)-Clavibacter michiganensis subsp. insidiosus has not been reported in China yet. It is a destructive disease to the alfalfa, but the cultural methods for detection of Clavibacter michiganensis subsp. insidiosus was biological methods and ELISA. So it is urgent to establish an effective method to detect it. In the article a subgroup specific probe was designed based on 16S rDNA of Clavibacter michiganensis subspp., and it was used to detect 4 strains...

A very serious quarantine bacterium(on the A2 list of China)-Clavibacter michiganensis subsp. insidiosus has not been reported in China yet. It is a destructive disease to the alfalfa, but the cultural methods for detection of Clavibacter michiganensis subsp. insidiosus was biological methods and ELISA. So it is urgent to establish an effective method to detect it. In the article a subgroup specific probe was designed based on 16S rDNA of Clavibacter michiganensis subspp., and it was used to detect 4 strains of Clavibacter michiganensis subspp and 10 species of other species of bacteria. The results show that no signal was detected for the others except Clavibacter michiganensis subsp. insidiosus. Few contaminations occur because the whole detection process is finished in the contained tubes, so the method is more specific than normal PCR. We founded a sensitive method to detect Clavibacter michiganensis subsp. insidiosus immediately and directly.

苜蓿萎蔫病菌是我国对外检疫性二类有害生物 ,目前国内尚无发生。在出入境检验检疫中主要是采用生物学和血清学方法进行检测 ,劳动强度大 ,耗费时间长。根据苜蓿萎蔫病菌与其它细菌菌株 1 6SrDNA序列差异 ,设计出对苜蓿萎蔫病菌具有稳定点突变特异性探针 ,利用该探针对棒形杆菌属 4个种及其它属细菌进行了实时荧光PCR检测实验。结果表明 ,只有苜蓿萎蔫病菌能检测到荧光信号 ,其它细菌没有荧光产生。该方法特异性强 ,灵敏度高 ,能检测到 2 1 4fg质粒DNA ,比常规PCR灵敏 1 0 0倍 ,而且整个过程只需要 2~ 3h。该方法可有效地应用于进出境病原菌检测之中。

 
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