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.

荧光定量PCR仪光学参数校准方法研究

荧光定量PCR仪性能参数主要包括温度参数和光学参数,其中温度参数校准较为成熟,而光学参数校准方法种类较多。本文对荧光定量PCR仪光学参数校准的DNA质粒标准物质法、染料标准物质法和温度光学物理装置法进行介绍和分析,并提出结合荧光定量PCR仪多通道荧光检测特点,开展对其多通道光学校准的物理校准方法研究。

荧光定量PCR仪多通道光学参数校准与结果分析

近年来,多重荧光PCR法广泛应用于肿瘤检测、动植物检疫、基因分型和物种鉴定等领域,但荧光定量PCR仪多荧光通道及免干扰设计会直接影响多重PCR检测结果。本文结合多重荧光PCR法的应用和荧光定量PCR仪多通道荧光检测能力设计,提出一种荧光定量PCR仪多通道光学参数校准方法,并通过实验,对荧光定量PCR仪的5个通道同时进行光学校准。实验证明:该方法具有可行性。

qPCR仪T_(m)校准的影响因素及结果分析

qPCR仪熔解曲线校准在物理参数层面,可更加直观地反应荧光定量PCR仪温控及光学系统的可靠性及相互影响,是全面评估荧光定量PCR仪的校准手段。由于熔解过程中,预熔解至熔解阶段的温度爬升速率不同,导致校准结果差异,因此,本文通过实验设计,分析了不同熔解爬升速率条件下Tm校准结果的差异和熔解曲线校准法的注意事项,为qPCR仪综合物理参数校准提供了参考。

荧光定量PCR仪的边缘效应与实验误差分析

目的:探讨荧光定量PCR分析仪边缘效应及实验误差的原因。方法:通过比较部分国外知名品牌荧光定量PCR仪的光路系统及加热模块设计原理,分析边缘效应及实验误差产生的原因。结果:传统荧光定量PCR仪光路设计缺陷和加热模块的孔间温度均一性不足会导致边缘效应和实验误差,是影响荧光定量PCR结果重复性的重要原因。结论:了解不同品牌荧光定量PCR仪的检测原理,选购性能优良的荧光定量PCR仪,尽量避免实验误差,以保证实验结果的准确性。

2种荧光定量PCR扩增仪检测结果的比较

目的:观察2种荧光定量PCR扩增仪测定乙肝病毒定量(HBV DNA)的结果是否一致。方法:用2台PCR扩增仪同时对20份标本进行检测,观察2台荧光定量PCR定量扩增仪对HBV DNA测定结果的差异。结果:对20份标本HBV DNA含量的对数值进行相关性分析,二者相关性良好(r=0.96)。结论:通过2种荧光定量PCR扩增仪对20份随机样本的检测结果比较,2种仪器检测结果具有较好的一致性,可用于同时检测HBV DNA。

实验室认可中两台荧光定量PCR仪的比对性研究

目的对ABI-7300荧光定量PCR仪和上海宏石SLAN96P荧光定量PCR仪进行比对和偏差分析。方法在本院检验科ISO15189实验室认可中使用ABI7300和宏石SLAN96P荧光定量PCR仪同时检测20例涵盖整个仪器线性的HBVDNA标本,以ABI-7300荧光定量PCR仪作为参比仪器,SLAN96P荧光定量PCR仪作为实验仪器,进行回归分析,计算偏倚%,评价其是否可接受。结果 ABI-7300荧光定量PCR仪与SLAN96P荧光定量PCR仪检测HBV-DNA的定量结果相对偏倚为6.9%,符合CNAS-CL36《医学实验室质量和能力认可准则在基因扩增检验领域的应用说明》明确要求即系统误差绝对值小于7.5%。结论 ABI-7300荧光定量PCR仪与SLAN96P荧光定量PCR仪检测的HBV-DNA结果具有可比性,可为临床提供可接受的检验结果。

荧光定量PCR仪光学校准方法与结果分析

实时荧光定量PCR仪特异性更强,自动化程度更高,且有效地解决了PCR污染的问题,应用领域及应用量都不断增加。但其设计更为复杂,温度模块和光学系统设计同时影响其性能和实验准确性,为定量PCR仪校准带来了巨大挑战。采用生物试剂等方式对定量PCR仪荧光部分校准缺乏溯源性,无法分析误差来源,存在较大缺陷。本文在对定量PCR仪影响因素和校准现状分析的基础上,采用Cyclertest 3D optical定量PCR仪光学校准系统对ABI 7500 Fast Real-Time定量PCR仪的温场部分和荧光系统进行了检测并对检测结果进行了分析,结果表明对温度模块和光学系统共同进行检测并分析相关性能够更科学全面地评估定量PCR仪性能,满足定量PCR仪校准需求。

实时荧光定量PCR仪用于流感病毒检测的效果分析

目的:探讨流感病毒检测中应用实时荧光定量PCR仪的价值。方法:选取2022年1月~2023年12月本中心采集检测的129份急性上呼吸道感染者的咽拭子标本展开研究,采集标本后实施实时荧光PCR仪、胶体金法测定标本中流感病毒核酸,分析两种方案检出情况、诊断效能。结果:实时荧光定量PCR仪阳性率19.38%,胶体金法阳性率为14.73%,实时荧光定量PCR仪检测阳性率高于胶体金法(P<0.05)。实时荧光定量PCR仪特异度(99.02%)、灵敏度(92.59%)、准确率(97.67%)高于胶体金法(P<0.05)。结论:在流感病毒检测中应用实时荧光定量PCR仪准确率、敏感度较高,且该方案可以迅速且有效检测出流感病毒核酸,为迅速地采取有效的疫情防控措施提供依据。

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.

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.

Analysis of Gene Expression of Seven Isoforms of ADP-glucose Pyrophosphorylase in Rice Endosperm under Different Temperature Conditions

[Objective] This study aimed to analyze the effects of temperature on the expression of AGPase isoform genes in rice endosperm during milk stage. [Method] Different temperature treatments （33 and 25 ℃ of daily mean temperature for high and normal temperature treatments, respectively） and the real-time fluorescence quantitative PCR （ FQPCR） were used to analyze the expression patterns of seven isoforms （AGPS1, AGPS2a, AGPS2b, AGPL1, AGPL2, AGPL3 and AGPL4） of ADPglucose pyrophosphorylase （AGPase） which was the key enzyme in starch synthesis and metabolism in rice endosperm of two rice varieties Teqing and Thai Fragrant Rice. [Result] The AGPase isoforms AGPS2b, AGPL2 and AGPL3 had much higher expression than the other four isoforms, thus they were thought to be the main expression patterns of AGPase in rice endosperm. The relative expressions of AGPL2 was the highest among all the isoforms. The relative expressions of AGPS2b, AGPL2 and AGPL3 were higher in the normal temperature treatment than in the high temperature treatment in both rice varieties. The relative expression of the three enzyme genes in milk stages in Teqing was higher than those in Thai Fragrant Rice under different temperature treatments. [Conclusion] This study provides a theoretical basis for further use of molecular biology techniques to cultivate stable high-quality rice varieties.