Detection of Ratoon Stunting Disease in Virus-free Seedcane via Real-time Fluorescence Quantitative PCR

This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease （RSD） in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from six plants of sugarcane ROC22, which had been confirmed RSD-positive by detecting the sugarcane juice, by employing the sugarcane seedlings production protocol. Real-time fluorescence quantitative PCR was used to detect RSD pathogens in tissue culture sam- pies. The results showed that target fragment of RSD pathogens was not found in all 10 samples in real-time fluorescence quantitative PCR, with the Ct values of 37 - 39. The healthy tissue culture sugarcane seedlings do not carry RSD pathogens, indicating that adopting healthy seedcane seedlings production technique could thoroughly get rid of RSD pathogens.

Real-time fluorescent quantitative PCR非特异性扩增的研究进展

Real-time fluorescent quantitative PCR(RT-qPCR)因其高效快速、操作简便、高度敏感等优点获得广泛应用,但因其强大的放大功能而容易出现非特异性扩增的问题,尤其是荧光染料法中更加明显。文章对RT-qPCR的原理、非特异性扩增的发生因素、解决方案及该技术的应用前景进行了综述。

Application of Real-time Fluorescent Quantitative PCR in Plant

Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.

Development and Preliminary Application of SYBR Green I Real-Time Fluorescence Quantitative PCR Method for Detecting Porcine Parvovirus Virus

According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 （NC001718） available in GenBank （NC_001718）, a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.

Detection of Lactobacillus acidophilus in Fermented Material by Real-time Fluorescent Quantitative PCR

The species distinctive PCR primer of Lactobacillus acidophilus （ L. acidophilus） was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative PCR （RT-PCR）. Analysis on RT-PCR results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-PCR method in the test. indicating that the established RT-PCR method could be aookued to the detection of L. acidophilus in fermented material.

Application of Real-time Fluorescent Quantitative PCR in Studies on Plants

Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.

Establishment of a Real-time Fluorescent Quantitative PCR Assay for Detection of Genetically Modified Maize Line MON88017

In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value （ 1. 541% ） was close to the actual value （ 1.50% ） and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.

Primary application of a real-time quantitative polymerase chain reaction for the detection of human breast cancer related novel gene-Metadherin expression

Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.

Development of real-time PCR method for rapid detection and quantification of Heterosigma akashiwo

To rapidly detect the harmful algae H.akashiwo qualitatively and quantitatively, sequences of the 18S rDNA deduced from H.akashiwo were used for designing species-specific primers, and a RFQ-PCR (Real-time Fluorescent Quantitative Polymerase Chain Reaction) method was developed for quantitative detection of H.akashiwo. Primer H.akashiwo and TaqMan probe were designed, and the specificity of primer was checked with PCR. A calibration curve was constructed with cycle threshold value against visual counted cell number. And the value of the curve was tested with other H.akashiwo samples, which were assayed with both the RFQ-PCR method and visual count under microscope.

基于芯片数据的烟草qRT-PCR内参基因鉴定与验证

实时荧光定量PCR(qRT-PCR)结果的准确性关键在于选择合适的稳定内参基因,而常用持家基因的表达不是恒定不变的,无法满足qRT-PCR准确定量的要求。为发掘烟草中优于传统持家基因的新内参基因,分析了烟草100张基因芯片数据,鉴定了稳定表达的候选内参基因,并利用烟草各个发育时期的RNA样品,通过qRT-PCR实验比较并验证了其表达的稳定性。结果显示,新鉴定的烟草内参基因HSC70-1表达稳定性优于所有的常用持家基因,HSC70-1,L25,EF-1α和HIST2H3A为烟草qRT-PCR实验中的最优内参基因组合,同时使用4个基因进行校正和标准化获得了可靠的定量结果。

QF-PCR与CMA技术在稽留流产及细胞学检查中的联合应用

目的探讨染色体微阵列芯片技术(CMA)和定量荧光PCR技术(QF-PCR)在稽留流产物及羊水细胞遗传学检查中的应用价值与方法学评价。方法回顾性分析2018年11月至2020年2月珠海市妇幼保健院妇科门诊169份稽留流产的绒毛组织及产前诊断中心进行孕中期羊膜腔穿刺的224例孕妇的临床资料,所有孕妇同时接受QF-PCR及CMA两种方法进行病因诊断,比较两种技术在非整倍体数目异常及结构异常诊断中的符合度。结果CMA分析169例流产绒毛,胎儿染色体异常检出119例(70.4%),其中数目异常为107例(89.9%),结构异常12例(10.1%),其中13、18、21三体及性染色体非整倍体异常在QF-PCR中检出率为100%,224例羊水细胞中QF-PCR检出21三体39例,18三体6例,13三体1例,46,XO 6例,47,XXX 4例,47,XXY 13例,47,XYY 4例,1例疑为45,XO嵌合体。检出5种常见染色体异常74例(33.0%)。以上病例均在CMA成功检出,CMA检出微变异150例(67.0%),7例拷贝数纯合状态(CNV AOH),2例无法有效识别异源性单亲二倍体(hetero UPD),检出率为99.1%。结论QF-PCR技术可快速诊断常见染色体非整倍体数目异常,可有效检出45,XO及47,XXY等性染色体拷贝数异常,检出率达到100%。但其存在局限性,只能检出大于20%的嵌合体,对于染色体存在的结构异常无法检出。CMA技术对小片段CNV的检测具有更高的检出能力,为基因型与表型的相关性研究提供依据并对夫妻双方再生育进行遗传风险评估和指导。

Occludin表达与胃癌发生发展的相关性

目的:探讨Occludin mRNA、蛋白表达水平与胃癌发生发展、侵袭转移的关系。方法:应用组织芯片免疫组化染色和实时荧光定量PCR的方法,检测occludin在胃腺癌组织中的表达并分析其与临床特征的相关性。结果:免疫组织化学检测显示,Occludin在癌旁正常胃黏膜上皮中阳性表达率高于胃腺癌组织(96.9%vs85.4%,84.6%,89.2%,78.5%)(P<0.05);Occludin的表达水平与胃腺癌TNM分期和分化程度有关(P<0.05)。实时荧光定量PCR的检测结果显示,Occludin mRNA在胃癌组织和癌旁正常胃组织中的表达无明显差异(P>0.05)。结论:Occludin蛋白的表达水平与胃腺癌的发生发展和侵袭转移呈负相关。

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

用原代细胞培养和人全基因表达谱芯片筛选鼻咽癌差异表达基因

目的利用细胞的原代培养技术及人全基因表达谱芯片技术初步筛选出鼻咽癌差异表达基因,以发现与鼻咽癌发生、发展相关的新的候选基因。方法分别用1例鼻咽癌细胞株C666及5例鼻咽癌培养出的原代细胞和3例鼻咽正常上皮的原代细胞,进行基因表达谱分析,并以荧光定量PCR及免疫组织化学验证芯片结果。结果原代培养的鼻咽癌细胞和鼻咽正常上皮细胞通过细胞角蛋白的免疫细胞化学染色、EBER1原位杂交和EBV-DNA荧光定量PCR证实;鼻咽癌原代细胞和鼻咽正常上皮原代细胞的基因表达谱有显著差异;4例以上共同表达的差异基因有493个,包括上调基因264个,下调基因229个。荧光定量PCR及免疫组织化学结果和芯片结果一致。结论用原代培养细胞及人类全基因组基因芯片构建基因表达谱能够准确、高效地筛选出鼻咽癌相关的差异表达基因,该基因表达谱的建立为研究鼻咽癌分子生物学机制、鼻咽癌的分子分型和分子诊断提供了重要的分子依据。

MaASR1基因通过乙烯途径提高拟南芥抗旱性的作用机制

香蕉MaASR1基因在植物响应逆境胁迫时发挥着重要作用,为了进一步研究MaASR1基因转入拟南芥后增强其抗旱性的分子机制,运用DNA芯片技术来筛选野生型拟南芥和转MaASR1基因拟南芥在不做任何胁迫处理和干旱胁迫处理条件下的差异基因。发现MaASR1提高拟南芥抗旱性与乙烯信号途径有密切的关系,对基因芯片中与乙烯途径相关的上调和下调大于2倍的差异基因进行了荧光定量PCR的验证。结果表明在干旱胁迫条件下,MaASR1的转入通过提高At ACS6和At ACO1的表达水平提高了拟南芥体内的乙烯合成水平。MaASR1的转入可以通过正调控乙烯反应和提高ERF类基因的表达来赋予植物抗旱性。以上结果为解析MaASR1基因作为转录因子通过乙烯途径提高植物抗旱能力的分子机制奠定了基础。

早孕小鼠胚胎着床前后子宫内膜microRNAs差异表达

应用microRNAs(miRNAs)芯片筛选早孕小鼠胚胎着床前后子宫内膜差异表达miRNAs,进行实时荧光定量PCR验证,运用靶基因预测数据库预测差异表达miRNAs调控的靶基因,为研究其在早孕小鼠胚胎着床过程中的作用打下基础。miRNAs芯片结果显示:孕6天比孕4天上调miRNAs有17个;下调miRNAs有18个。实时荧光定量PCR结果显示芯片数据可靠。通过miRGen、Targetscan、Pictar等数据库筛查,初步获得差异表达的部分miRNAs多指向与凋亡和抗凋亡相关的靶基因。表明miRNAs可能在胚胎着床前后子宫内膜中起重要的调控作用。

Changes of the Transcriptional Levels of Molecules Associated with Endogenous Antigen Processing and Presentation in Porcine Skin-derived Dendritic Cells Infected with PCV2 in vivo

[Objective] This study aimed to investigate the changes of the transcriptional levels of molecules associated with endogenous antigen processing and presenta- tion in porcine skin-derived dendritic cells infected with PCV2 in vivo. [Method] Healthy 40-day-old Landrace piglets were infected with porcine circovirus type 2 （PCV2） and euthanized on the 34, 7rd, 14th, 21st and 35th d post inoculation （DPI）. The porcine skin-derived dendritic cells （DCs） were collected to analyze the transcrip- tional levels of molecules （LMP7, UBP, MHC-I, calreticulin） associated with endogenous antigen processing and presentation by using real-time fluorescent quantitative PCR （real-time FQ-PCR）. [Result] The results showed that the level of LMP7 mR- NAs was reduced significantly on the 3DPI （P〈0.05）; the level of UBP mRNAs was consistently up-regulated, which increased significantly on the 21DPI and 35DPI （P〈 0.05）; the level of MHC-I mRNAs was significantly down-regulated on the 7DPI （P〈 0.05）; the level of calreticulin mRNAs was up-regulated slightly without significant dif- ference. [Conclusion] PCV2 can inhibit the endogenous antigen processing and presentation ability of porcine skin-derived DCs at early stages of infection.

胎儿染色体异常快速产前检测方法的研究进展

快速产前检测方法是产前诊断的重要组成部分,包括细胞和分子遗传学和超声诊断。由于细胞和分子遗传学检测技术的介入,胎儿染色体非整倍体的快速检出及某些染色体结构异常的鉴别能力,得到了很大的提高。目前临床上较常用的细胞和分子遗传技术包括荧光原位杂交技术(FISH)、荧光定量PCR(QF-PCR)、多重连接探针扩增技术(MLPA)和微阵列比较基因组杂交技术(array-CGH)。在怀孕的第1和第2个"3个月"阶段进行有效的超声检查,常见的染色体异常都可以被检测出来。因此,将细胞和分子遗传技术与超声检测相结合,可以提高胎儿染色体非整倍体的检出率和产前诊断的准确性,以减少缺陷婴儿的出生。

小脑变性相关蛋白2 mRNA在癫痫患者脑组织中的表达及其临床意义

目的了解小脑变性相关蛋白2(CDR2)mRNA在癫痫(EP)患者脑组织中的表达及其临床意义。方法将48例EP患者分成耐药(40例)和非耐药(8例)两组,用基因芯片扫描和FQ-PCR分别检测脑组织中CDR2基因表达,并与对照组比较。结果基因芯片扫描提示CDR2 mRNA在EP患者与对照组中的表达差异有统计学意义(P<0.05)。FQ-PCR结果与基因芯片扫描一致。结论EP患者脑组织中有CDR2 mRNA表达,由于其表达产物可通过血-脑脊液屏障进入脑脊液(CSF)和血液中,因而检测CDR2 mRNA在血或CSF中的表达产物有可能成为诊断EP的一个重要指标。

应用SNP array检测流产物三倍体

目的染色体微阵列技术(CMA)使用的基因芯片一般为SNP array、array CGH,分别使用这两种基因芯片对流产物进行CMA检测,对比两种芯片对流产物三倍体检测的准确性。方法应用SNP array基因芯片,对2016年6月至2017年10月在广东省妇幼保健院就诊397例患者的流产物进行SNP array检测;并对SNP array结果为三倍体的行定量荧光PCR(QF-PCR)验证;对69,XXY和69,XXX流产物用array CGH进行检测。结果SNP array检测出8例三倍体,3例69,XXY,4例69,XXX,1例亚三倍体68,XX;SNP array检测结果与QF-PCR结果一致;array CGH不能检测出流产物三倍体。结论 SNP-array相对array CGH能有效检出流产物三倍体,减少误诊、漏诊,对评估再生育风险及遗传咨询有重要意义。