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P72、MGF和CD2v基因的非洲猪瘟三重荧光定量PCR检测方法的建立

非洲猪瘟在临床上出现多种毒株,主要分为野毒株和基因变异毒株。因其在临床上造成不同程度的危害,有必要建立一种方法进行正确诊断和健康监测。为实现野毒株和基因变异毒株的鉴别诊断,研究根据GenBank中的ASFV中国流行株(登录号:MK333180.1)的P72、CD2v、MGF基因保守序列设计引物,建立一种可以同时检测上述三种基因的三重荧光定量PCR方法。三重荧光定量PCR方法对猪细小病毒(PPV)、猪伪狂犬病病毒2型(PRV)和猪圆环病毒(PCV2)检测均无交叉反应,表现出较强的特异性。敏感性检测显示针对P72、CD2v和MGF重组质粒标准品的最低检测下限为103~102 copies/mL。在重复性试验中,大部分组间变异系数均小于2%或接近2%,具有较好的重复性。试验表明,三重荧光定量PCR方法可用来鉴别野毒株和基因变异毒株。

基于全自动核酸提取的荧光定量PCR鉴别羊肉制品真伪

目的建立一种基于自动化的快速核酸提取方法,联合三重实时荧光定量PCR技术,鉴别羊肉制品的真伪。方法分别采用全自动核酸提取法、磁珠手提法与柱提法对不同状态羊肉制品进行核酸提取,比较不同方法的提取时间差异,通过三重实时荧光定量PCR扩增结果评估不同方法的提取效果。以GB/T38164—2019《常见畜禽动物源性成分检测方法实时荧光PCR法》为对照,对市售的羊肉制品进行平行检测,评估本方法的检测性能。结果与磁珠手提法、柱提法相比,全自动核酸提取法的提取时间缩短了15~30 min,且提取的核酸扩增效果更好。全自动核酸提取法在羊猪混合样品中羊源核酸检出限为0.010mg/g。全自动核酸提取法在生鲜肉、生肉及熟肉制品等多种类型产品中提取的羊源核酸PCR检测结果的变异系数(coefficient of variation,CV)均小于3.000%。分别采用三重实时荧光定量PCR法与GB/T38164—2019对全自动提取的21份市售的涵盖不同类型的羊肉制品核酸进行平行检测,符合率为100.00%。结论本研究方法可为羊肉制品掺假的快速真伪鉴别提供操作简便、稳定灵敏、快速准确的整套解决方案。

诺如病毒和轮状病毒三重荧光定量PCR检测方法的建立

为了建立一种能同时检测GI型诺如病毒(GI-NV)、GII型诺如病毒(GII-NV)和A群轮状病毒(A-RV)的三重荧光定量PCR检测方法,本试验根据GI-NV和GII-NV的开放阅读框1(Orf1)与开放阅读框2(Orf2)基因之间保守序列、A-RV的非结构蛋白3(Nsp3)基因保守序列,分别设计合成3对特异性引物和3个TaqMan探针,人工合成含有目标序列的基因,制备重组质粒标准品,绘制标准曲线,并对该检测方法的特异性、灵敏度、重复性进行评估。同时通过检测收集的109份动物源食品样品,对建立的方法与商品化试剂盒进行检测比对。结果显示:该方法可以特异性地区分GI-NV、GII-NV和A-RV,而与其他病原无交叉反应;该方法的最低检出限均为1×102 copies/μL的质粒标准品,具有较高的敏感性;组内和组间重复性试验结果显示,变异系数均小于5%,重复性较好;该方法与商品化的试剂盒检测结果符合率为100%。结果表明,本试验建立的检测方法能够快速、准确区分GI-NV、GII-NV和A-RV。

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.

Analysis on Tissue-specific Expression of Dp XTH1 and Dp XTH2 Genes in Dahlia

[Objective] This study aimed to investigate the functions and related mechanisms of xyloglucan Endotransglycosylase/hydrolases （XTHs） during the growth and development of dahlia. [Method] Using /3-actin as the reference gene, the rela- tive transcription levels of DpXTH1 and DpXTH2 genes in roots, stems, leaves and petals of dahlia were analyzed by real-time RT-PCR. [Result] The DpXTH1 and DpXTH2 were not expressed in the roots, but expressed abundantly in the petals of dahlia. There were little expressions in the stems and leaves of dahlia. [Conclusion] The DpXTH1 and DpXTH2 were petal-specific genes and closely related to the growth and development of petals in dahlia.

基于基因拷贝数的缺失型α地中海贫血检测体系研究

目的建立并评价基于α珠蛋白基因拷贝数变化的缺失型α地中海贫血检测体系。方法建立三重TapMan实时荧光定量PCR检测体系,结合2^-△△ct相对定量分析的原理,根据缺失型α地中海贫血分子机制中α珠蛋白基因和看家基因的拷贝数比例差异,快速诊断α地中海贫血基因拷贝数。以本研究建立的方法检测69例已知α地中海贫血基因型标本和100例临床标本,并通过Gap-PCR+MLPA技术进行验证,评价该方法的敏感度与特异度。结果本研究建立的检测体系对100例临床标本进行α地中海贫血基因型的检测,敏感度和特异度均为100%;临床常规检测方法的特异度为100%,敏感度只有96%。结论本研究建立的检测体系敏感度高,特异性好,能够检测临床常规检验方法未检出的非常见型α地中海贫血基因型,减少或避免出生缺陷,优生优育。

Efflux pump gene hefA of Helicobacter pylori plays an important role in multidrug resistance

AIM: To determine whether efflux systems contribute to multidrug resistance of H pylori. METHODS: A chloramphenicol-induced multidrug resistance model of six susceptible H pylori strains (5 isolates and H pylori NCTC11637) was developed. Multidrug-resistant (MDR) strains were selected and the minimal inhibitory concentration (MIC) of eryth-romycin, metronidazole, penicillin G, tetracycline, and ciprofloxacin in multidrug resistant strains and their parent strains was determined by agar dilution tests. The level of mRNA expression of hefA was assessed by fluorescence real-time quantitative PCR. A H pylori LZ1026 knockout mutant (ΔH pylori LZ1026) for (puta-tive) efflux protein was constructed by inserting the kanamycin resistance cassette from pEGFP-N2 into hefA, and its susceptibility profiles to 10 antibiotics were evaluated. RESULTS: The MIC of six multidrug-resistant strains (including 5 clinical isolates and H pylori NCTC11637) increased signifi cantly (≥ 4-fold) compared with their parent strains. The expression level of hefA gene was significantly higher in the MDR strains than in their parent strains (P = 0.033). A H pylori LZ1026 mutant was successfully constructed and the ΔH pylori LZ1026 was more susceptible to four of the 10 antibiotics. All the 20 strains displayed transcripts for hefA that con-fi rmed the in vitro expression of these genes.CONCLUSION: The efflux pump gene hefA plays an important role in multidrug resistance of H pylori.

Temperature Stress at Grain Filling Stage Mediates Expression of Three Isoform Genes Encoding Starch Branching Enzymes in Rice Endosperm

An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures （32℃ for high temperature and 22℃ for optimum temperature） at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes （SBEI, SBEIII and SBE/V） encoding starch branching enzyme （SBE） in the endosperms were studied by the real-time fluorescence quantitative PCR （FQ-PCR） method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.

Expressions of Toll Like Receptor(TLR)Genes in Paralichthys olivaceus After Induced by Different Extracts of Edwardsiella tarda

Toll like receptors(TLRs)are the main innate immune‘pattern recognition receptors’of animals,which play a central role in host cell recognition and responses to invasive pathogens,particularly common structures of microbial pathogens.In this study,the gene expression profiles of TLRs in the spleen,head kidney,gill,small intestine,liver,muscle,and heart of healthy Paralichthys olivaceus were detected by real-time quantitative PCR(qPCR).The TLR family members were widely expressed in different tissues with different basic expression profiles.The highest expressions of TLR1,5m,7,8,9,14,and 21 were found in the spleen;the highest expressions of TLR3 and TLR21 were found in the gill;the highest expressions of TLR2 and 5s were found in the small intestine.The second highest expressions of TLR3,7,and 8 were found in small intestine.The gene expression profiles of TLRs stimulated with Edwardsiella tarda DNA,RNA,and lipopolysaccharide(LPS)were also detected in spleen,head kidney and gill.TLR9 and TLR21 were sensitive to E.tarda DNA;TLR 8 and TLR21 were sensitive to E.tarda RNA;and TLR1 and TLR14 were sensitive to E.tarda LPS.The expressions of the other TLR genes showed no significant changes.The results imply that the expressions of these TLR genes in P.olivaceus are differently regulated in the whole body and play important roles in the immune response against E.tarda infection.