A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA（rDNA） and internal transcribed spacer（ITS）.A pair of primers PBF1/PBR1 was designed based on the conse...A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA（rDNA） and internal transcribed spacer（ITS）.A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.展开更多
Objective:To evaluate the antiviral activity of the alcohol extract of Styela plicata on DHBV (duck hepatitis B virus) in vivo. Methods: Guangzhou-Sheldrake ducklings congenitally infected with DHBV were assigned to r...Objective:To evaluate the antiviral activity of the alcohol extract of Styela plicata on DHBV (duck hepatitis B virus) in vivo. Methods: Guangzhou-Sheldrake ducklings congenitally infected with DHBV were assigned to receive the alcohol extract of Styela plicata or lamivudine for 30 consecutive days. The DHBV DNA of sera was detected by RT-PCR. and the histological analysis of duckling liver was evaluated. Results:Thirty days after therapy,histological analysis of duckling liver showed that the ducklings receiving the alcohol extract of Styela plicata or lamivudine exhibited catabatic status in the degree of liver cell degeneration and inflammation compared with the ducklings receiving normal diet. DHBV DNA of sera from alcohol extract of Styela plicata-treated ducklings and lamivudine-treated ducklings all produced significantly lower levels compared with ducklings receiving normal diet (P<0. 01 ). Although these treatment groups all exhibited a rebound phenomenon 10 d after withdrawal of medication, they still exhibited a significant lower level of serum DHBV DNA compared with the control group responded to normal diet (P<0. 05, P<0. 01). Conclusion:Styela plicata may be an effective antiviral medicine in treating chronic hepatitis B. The data of this experiment will be valuable in studying the therapeutic role and the potential therapeutic mechanism of Styela plicata.展开更多
【目的】建立基于鸡毒支原体种特异性粘附蛋白编码基因pvpA的实时荧光定量PCR检测方法。【方法】根据GenBank公布的不同国家和地区鸡毒支原体pvpA基因序列,在高度保守区域设计一对引物。以临床分离鸡毒支原体RC1为模板扩增pvpA基因,将...【目的】建立基于鸡毒支原体种特异性粘附蛋白编码基因pvpA的实时荧光定量PCR检测方法。【方法】根据GenBank公布的不同国家和地区鸡毒支原体pvpA基因序列,在高度保守区域设计一对引物。以临床分离鸡毒支原体RC1为模板扩增pvpA基因,将其连接到PMD19-T载体,转化至大肠杆菌DH5α中,经PCR和酶切鉴定并测序验证后得到阳性重组质粒rPvpA90。以rPvpA90为模板建立SYBR Green I荧光定量的标准曲线和溶点曲线,并进行特异性,灵敏性,重复性及临床样本检测试验,评价该方法的可行性。【结果】所建立的荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系,溶点曲线特异,相关系数为0.990。最低检测限为72拷贝/20μL,其敏感性比常规PCR至少高100倍;无论是对不同病原DNA单模板还是几种病原DNA混合模板进行扩增,该方法都呈现很好的特异性;重复性试验中,批内和批间变异系数均小于2%,表明该方法重现性好;临床样本的检测结果表明所建立的荧光定量PCR检测方法的检测率明显高于常规PCR方法。【结论】本研究初步建立了基于种特异性基因pvpA的鸡毒支原体荧光定量PCR方法,为养禽场诊断和监测鸡毒支原体病原提供一种新的特异、灵敏的方法。展开更多
该工作以富含大量胸腺嘧啶（Thymine，T）核酸单链为识别分子，SYBR Green I （SG）为荧光基团，建立了一种简单、灵敏的荧光增强法检测Hg2＋。由于T-Hg2＋-T键的形成，富T单链自我折叠或者两两配对形成双链DNA结构，当溶液中的SG嵌入DN...该工作以富含大量胸腺嘧啶（Thymine，T）核酸单链为识别分子，SYBR Green I （SG）为荧光基团，建立了一种简单、灵敏的荧光增强法检测Hg2＋。由于T-Hg2＋-T键的形成，富T单链自我折叠或者两两配对形成双链DNA结构，当溶液中的SG嵌入DNA双链中时，SG荧光强度显著增强。实验结果表明，SG荧光强度随着Hg2＋浓度的增加而增加。在最优实验条件下，SG的荧光强度与Hg2＋的浓度在4．000×10-7~2．000×10-6 mol/L范围内呈线性关系，检出限为3．900×10-8 mol/L。该方法在含5．0％湘江水实际样品中获得的回收率为98．72％~104．5％，因此该传感器可用于实际湘江水样品中Hg2＋的测量。展开更多
为了建立一种可以快捷、灵敏、安全地检测D型流感病毒(Influenza D virus,IDV)的实时荧光定量PCR(quantitative real time PCR,RT-qPCR)方法,试验首先将IDV NP基因作为检测目标,根据其序列的保守区设计了一对RT-qPCR引物,然后采用单一...为了建立一种可以快捷、灵敏、安全地检测D型流感病毒(Influenza D virus,IDV)的实时荧光定量PCR(quantitative real time PCR,RT-qPCR)方法,试验首先将IDV NP基因作为检测目标,根据其序列的保守区设计了一对RT-qPCR引物,然后采用单一变量法对RT-qPCR反应条件中的引物浓度和退火温度进行优化,建立了一种可检测IDV的RT-qPCR方法,并对该方法的灵敏度、重复性和特异性进行检测,最后应用该方法进行临床样本检测。结果表明:所建立的RT-qPCR方法的最佳引物浓度和退火温度分别为400 nmol/L和61℃,标准曲线为y=-3.572x+42.02(R^(2)=0.9993);该方法能检测到的最低拷贝数为8.30×10^(1)copies,灵敏度是常规PCR的100倍左右;组内重复和组间重复变异系数(CV)值均小于3%;同时检测牛呼吸道合胞体病毒(BRSV)、牛传染性鼻气管炎病毒(IBRV)、牛病毒性腹泻病毒(BVDV)、牛冠状病毒(BCoV)、牛副流感病毒3型(BPIV-3)、牛疱疹病毒4型(BHV-4)和牛流行热病毒(BEFV)的结果均为阴性;应用该方法对收集到的244份有呼吸道症状的牛鼻拭子样品进行检测,阳性率为1.23%。说明成功建立了IDV RT-qPCR检测方法,该方法具有灵敏度高、重复性和特异性好的特点,为IDV的防控提供了可靠的技术支撑。展开更多
基金supported by the emarked fund for Moden Agro-Industry Technology Research System, China (CARS25)the National Natural Science Foundation of China (31201473)the Key Laboratory of Biology and Genetic Improvement of Horticulture Crops, Ministry of Agriculture, China
文摘A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA（rDNA） and internal transcribed spacer（ITS）.A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.
基金Supported by the grants from the Social Development Program of Department of Science and Technology of Guangdong Province (2004B30101009).
文摘Objective:To evaluate the antiviral activity of the alcohol extract of Styela plicata on DHBV (duck hepatitis B virus) in vivo. Methods: Guangzhou-Sheldrake ducklings congenitally infected with DHBV were assigned to receive the alcohol extract of Styela plicata or lamivudine for 30 consecutive days. The DHBV DNA of sera was detected by RT-PCR. and the histological analysis of duckling liver was evaluated. Results:Thirty days after therapy,histological analysis of duckling liver showed that the ducklings receiving the alcohol extract of Styela plicata or lamivudine exhibited catabatic status in the degree of liver cell degeneration and inflammation compared with the ducklings receiving normal diet. DHBV DNA of sera from alcohol extract of Styela plicata-treated ducklings and lamivudine-treated ducklings all produced significantly lower levels compared with ducklings receiving normal diet (P<0. 01 ). Although these treatment groups all exhibited a rebound phenomenon 10 d after withdrawal of medication, they still exhibited a significant lower level of serum DHBV DNA compared with the control group responded to normal diet (P<0. 05, P<0. 01). Conclusion:Styela plicata may be an effective antiviral medicine in treating chronic hepatitis B. The data of this experiment will be valuable in studying the therapeutic role and the potential therapeutic mechanism of Styela plicata.
文摘【目的】建立基于鸡毒支原体种特异性粘附蛋白编码基因pvpA的实时荧光定量PCR检测方法。【方法】根据GenBank公布的不同国家和地区鸡毒支原体pvpA基因序列,在高度保守区域设计一对引物。以临床分离鸡毒支原体RC1为模板扩增pvpA基因,将其连接到PMD19-T载体,转化至大肠杆菌DH5α中,经PCR和酶切鉴定并测序验证后得到阳性重组质粒rPvpA90。以rPvpA90为模板建立SYBR Green I荧光定量的标准曲线和溶点曲线,并进行特异性,灵敏性,重复性及临床样本检测试验,评价该方法的可行性。【结果】所建立的荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系,溶点曲线特异,相关系数为0.990。最低检测限为72拷贝/20μL,其敏感性比常规PCR至少高100倍;无论是对不同病原DNA单模板还是几种病原DNA混合模板进行扩增,该方法都呈现很好的特异性;重复性试验中,批内和批间变异系数均小于2%,表明该方法重现性好;临床样本的检测结果表明所建立的荧光定量PCR检测方法的检测率明显高于常规PCR方法。【结论】本研究初步建立了基于种特异性基因pvpA的鸡毒支原体荧光定量PCR方法,为养禽场诊断和监测鸡毒支原体病原提供一种新的特异、灵敏的方法。
文摘该工作以富含大量胸腺嘧啶（Thymine，T）核酸单链为识别分子，SYBR Green I （SG）为荧光基团，建立了一种简单、灵敏的荧光增强法检测Hg2＋。由于T-Hg2＋-T键的形成，富T单链自我折叠或者两两配对形成双链DNA结构，当溶液中的SG嵌入DNA双链中时，SG荧光强度显著增强。实验结果表明，SG荧光强度随着Hg2＋浓度的增加而增加。在最优实验条件下，SG的荧光强度与Hg2＋的浓度在4．000×10-7~2．000×10-6 mol/L范围内呈线性关系，检出限为3．900×10-8 mol/L。该方法在含5．0％湘江水实际样品中获得的回收率为98．72％~104．5％，因此该传感器可用于实际湘江水样品中Hg2＋的测量。
文摘为了建立一种可以快捷、灵敏、安全地检测D型流感病毒(Influenza D virus,IDV)的实时荧光定量PCR(quantitative real time PCR,RT-qPCR)方法,试验首先将IDV NP基因作为检测目标,根据其序列的保守区设计了一对RT-qPCR引物,然后采用单一变量法对RT-qPCR反应条件中的引物浓度和退火温度进行优化,建立了一种可检测IDV的RT-qPCR方法,并对该方法的灵敏度、重复性和特异性进行检测,最后应用该方法进行临床样本检测。结果表明:所建立的RT-qPCR方法的最佳引物浓度和退火温度分别为400 nmol/L和61℃,标准曲线为y=-3.572x+42.02(R^(2)=0.9993);该方法能检测到的最低拷贝数为8.30×10^(1)copies,灵敏度是常规PCR的100倍左右;组内重复和组间重复变异系数(CV)值均小于3%;同时检测牛呼吸道合胞体病毒(BRSV)、牛传染性鼻气管炎病毒(IBRV)、牛病毒性腹泻病毒(BVDV)、牛冠状病毒(BCoV)、牛副流感病毒3型(BPIV-3)、牛疱疹病毒4型(BHV-4)和牛流行热病毒(BEFV)的结果均为阴性;应用该方法对收集到的244份有呼吸道症状的牛鼻拭子样品进行检测,阳性率为1.23%。说明成功建立了IDV RT-qPCR检测方法,该方法具有灵敏度高、重复性和特异性好的特点,为IDV的防控提供了可靠的技术支撑。
