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.

PCR技术在食品微生物检测中的应用研究

PCR技术因具有高灵敏度、高特异性和快速性的特点,在食品微生物检测领域广泛使用。本文阐述食品中常见的致病微生物、PCR技术的基本原理、食品中常见致病微生物的PCR检测方法,探讨PCR技术在食品微生物检测中的应用,并介绍PCR技术在食品微生物检测中的发展趋势。

应用TaqMan-MGB探针实时荧光定量PCR技术快速检测青海省海西州鼠疫自然疫源地鼠疫耶尔森菌耐链霉素基因

目的 应用TaqMan-MGB探针实时荧光定量PCR技术快速检测青海省海西州鼠疫自然疫源地鼠疫菌耐链霉素基因,为今后该地区突发人间鼠疫的精准临床用药提供理论依据。方法 分离培养海西地区1957—2009年间取自鼠疫患者、媒介昆虫及中间宿主的代表性鼠疫菌110株,提取其DNA,针对我国链霉素耐药基因rpsl基因设计引物P-F和P-R和TaqMan-MGB探针Probe1 [FAM]和Probe2[VIC],利用荧光定量PCR技术,进行耐药rpsl基因筛查。结果 110株被试菌株中FAM检测均为阳性(RFU峰值>2000);VIC阳性的为0株(RFU峰值<200)。阳性对照和空白对照成立。结论 实时荧光定量PCR结果显示,该地区未检测出耐链霉素菌株。

荧光定量PCR技术与酶联免疫法在手足口病肠道病毒检测中的应用价值比较

目的探讨荧光定量PCR技术(FQ-PCR)与酶联免疫法(ELISA)在手足口病肠道病毒检测中的应用价值。方法选取2021年1月至2022年12月就诊于广东省江门市妇幼保健院的疑似238例手足口病患儿,均行FQ-PCR检测与ELISA检测,统计FQ-PCR与ELISA病毒阳性检出率,以病毒分离培养及临床表现、血清实验室综合诊断结果为金标准,对比FQ-PCR与ELISA检测敏感度与特异度、准确度及检测窗口期。结果238例患儿中137例(57.56%)最终确诊为手足口病。FQ-PCR检测敏感度与特异度、准确度均高于ELISA,差异有统计学意义(P<0.05);FQ-PCR病毒阳性检出率较ELISA高,差异有统计学意义(P<0.05);FQ-PCR对通用型肠道病毒、Cox A16、EV71检测窗口期均短于ELISA检测,差异有统计学意义(P<0.05)。结论与ELISA相比,FQ-PCR在手足口病肠道病毒检测中具有更高的特异度、敏感度,可提高阳性检出率,且检测窗口期更短,利于临床早期实施治疗。

RAPID DETECTION OF ONCOGENE AMPLIFICATION IN PARAFFIN SECTIONS OF BREAST CARCINOMAS USING QUANTITATIVE DIFFERENTIAL PCR AND FLUORESCENT DNA TECHNOLOGY

Gene amplification is a common mechanism of oncogene activation and contributes to tumor progression. Analysis of such genetic alterations are relevant to the understanding of tumor genetics and could provide prognostic information for the individual patient. Standard analytical approaches using Southern blot and slot blot require a large amount of good

虚拟仿真在“生物技术药物学实验”中的应用

由于许多实验环境无法满足新冠病毒取样和检测的要求,且病原微生物新冠病毒具有强传染性和致病性,学生很难开展感染性实验操作。因此,将虚拟仿真技术与新冠病毒实时荧光定量PCR检测相结合构建了虚拟实验平台,其中涵盖实验导学、临床取样及实时荧光定量PCR检测等主要内容,重点模拟RNA提取、病毒序列获取与分析、引物设计、反转录、实时荧光定量PCR及结果分析等操作。通过虚拟环境,在保障学生安全的基础上,让学生更深入地了解新冠病毒实时荧光定量PCR的检测,大大提高了生物技术药物学实验教学的效率。

实时荧光PCR在乙肝病毒DNA检测中的临床意义

目的用实时荧光定量PCR检测乙型肝炎患者HBV DNA结果进行分析,以探讨其临床诊疗意义。方法对630例血清标本用时间分辨荧光免疫技术定量测定乙肝炎病毒血清学指标,用荧光定量PCR法检测血标本中的HBVDNA,并依据乙肝两对半结果进行归类分组。结果 630份标本中,大三阳206例标本中有173份HBV-DNA为阳性,阳性率为84.0%,其PCR定量拷贝数为(2.13±0.72)×107/ml;小三阳211例中有64份HBV-DNA阳性,阳性率达到30.3%,PCR定量拷贝数为(1.61±0.85)×106/ml;69份HBs Ab(+)、HBe Ab(+)、HBc Ab(+)标本有33份HBV-DNA阳性,阳性率47.8%;27份HBs Ag(+)、HBc Ab(+)标本中有8份HBV-DNA为阳性,阳性率为29.6%;22份HBe Ab(+)、HBc Ab(+)标本有3份PCR HBV-DNA阳性,阳性率13.6%;20份HBs Ag(+)、HBs Ab(+)、HBe Ag(+)、HBc Ab(+)标本中有17份HBV-DNA为阳性,阳性率为85.0%;17份HBs Ab(+)、HBe Ab(+)阳性标本有3份PCR HBV-DNA阳性,阳性率为17.6%;13份HBs Ab(+)、HBc Ab(+)标本有1份HBV-DNA阳性,阳性率7.7%;12份HBc Ab(+)标本有5份PCR HBV-DNA阳性,阳性率为41.7%;2份HBs Ab(+)的标本HBV-DNA均为阴性,PCR定量拷贝数为<1.00E×103;1份HBs Ag(+)标本HBV-DNA均为阳性,阳性率为100%;其余30例全阴性结果和各少见模式基本未见有HBV-DNA的检出。结论 PCR定量测定HBV-DNA可以真实反映体内乙肝病毒感染和复制及病毒载量情况,更有利于临床治疗和疗效观察。

荧光定量PCR技术在环境微生物检测中的应用

阐述了实时荧光定量PCR技术的原理、特点及其在环境微生物检测中的应用与研究进展,分析了该技术在环境微生物检测中存在的问题,并探讨了存在的问题,以及其发展和应用前景。

高灵敏荧光定量PCR技术在乙肝患者中的应用分析

目的研究高灵敏荧光定量PCR技术在乙肝患者中的应用效果。方法现研究选取的研究对象为2016年10月—2017年6月在我院进行治疗的乙肝患者923例和同期在本院进行体检的健康体检者200例。使用高灵敏荧光定量PCR技术检测两组受检者。结果不同血清标志物阳性乙肝患者的HBV-DNA拷贝数差异存在统计学意义(P<0.05),阳性率分别为35.97%、57.96%、6.07%,乙肝患者间进行比较差异具有统计学意义(P<0.05)。结论采用高灵敏荧光定量PCR技术可较好的诊出乙肝,还可监测治疗过程及评价药效。

荧光定量PCR技术在肺结核诊断中的应用研究

目的探讨荧光定量PCR技术在肺结核诊断中的应用价值。方法采用浓缩集菌涂片抗酸染色显微镜检查、美国BD960型分枝杆菌培养仪进行结核菌快速培养鉴定、荧光定量PCR结核分枝杆菌检测三种方法同时检测1 000例肺结核患者的痰液标本,比较检测结果。结果PCR技术诊断阳性276例,阳性率27.6%(276/1 000);结核菌快速培养阳性252例,阳性率25.2%(252/1 000);涂片抗酸染色显微镜检查172例,阳性率17.2%(172/1 000),结核抗酸菌涂片阳性率低于PCR技术及快速培养法(P<0.05),且荧光定量PCR技术与结核菌诊断金标准快速培养阳性符合率为91.3%(252/276)。结论荧光定量PCR技术检测结核分枝杆菌DNA技术,对结核病进行诊断,与传统的涂片、培养相比较,可提高肺结核诊断的敏感性、特异性和及时性,以期使肺结核患者得到早期诊断及有效治疗。

荧光定量PCR检测女性HPV-DNA的临床价值

目的:评价荧光定量PCR技术在女性生殖道人乳头状瘤病毒(HPV-DNA)中的检测价值。方法:收集2016年10月-2021年5月体检中心26630例健康体检者、门诊12521例及住院患者5576例的宫颈分泌物,采用PCR荧光探针法进行HPV-DNA检测,对检测结果进行统计分析。结果:门诊患者与住院患者HPV感染率均高于健康体检者,差异有统计学意义(P<0.05)。健康体检者、门诊及住院患者荧光定量PCR法检出HPV感染7860例,阳性率为17.57%;其中高危型2542例(32.34%)、低危型5318例(67.66%);与病理活检结果比较,荧光定量PCR法检测高危型HPV阳性符合率为93.30%,阴性符合率为93.59%,总符合率为93.55%。结论:荧光定量PCR法是HPV-DNA有效的临床检测技术,在检测常见的高危型HPV方面有较好结果,可用于女性大规模体检初筛。

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.