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.

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.

Development of Real-Time Fluorescent PCR for Rapid Detection of Haempohlius parasuis

[ Objective] To develop a real-time fluorescent PCR assay for rapid detection of Haempohlius parasuis （HPS）. [ Method] According to the conservative sequences of 16 S rRNA genes of HPS published in GenBank, a pair of specific primers was designed. The real-time fluorescent PCR was developed by optimizing primer concentration and annealing temperature. And its specificity and reproducibility were evaluated. Ten HPS- suspected samples were detected by the developed method. [ Result] The lowest detection limit of the developed real-time fluorescent PCR was 50 copies/μl. This method had good reproducibility, and its coefficient of variation was lower than 2%. Only HPS rather than Streptococcus suis type 2, Staphylococcus aureus, E. coli DH5 alpha, and swine Salmonella typhi could be detected by the developed real-time fluorescent PCR. The HPS-pesitive samples detected by this method were also positive when they were detected by isolation of bacteria or conventional PCR. [ Conclusion] The developed real-time fluorescent PCR is rapid, sensitive, specific and highly reproducible; thus, it can be used for rapid detection of HPS.

Synchronously Detecting Allergenic Ingredients of Peanut and Sesame in Food by Real-time Fluorescent PCR

Peanut,sesame and other raw materials of food are allergens for special populations.In this study,specific primers and TaqMan probes labeled by different fluorescences were designed targeting Ara h 2 gene of peanut and Ses i 1 gene of sesame.After the optimization of reaction conditions,a real-time fluorescent PCR method was established for simultaneous detection of allergenic ingredients of peanut and sesame in food.Genomic DNA samples of peanut,sesame,rice,wheat,barley,soybean,celery,maize,potato,tomato,walnut,groundnut in shell,cashew nut,sunflower seed,almond,apple,pear and strawberry,pork,beef,mutton and fish were used as templates for PCR amplification with deionized water as negative control template.Results indicated that the established real-time fluorescent PCR method could specifically identify allergenic ingredients of peanut and sesame simultaneously.Sensitivity test showed that the minimum detection limit of this method was 0.01%.Therefore,the established real-time fluorescent PCR method is a specific,sensitive and effective assay for simultaneously detecting allergenic ingredients of peanut and sesame in food.

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.

Effects of Different Temperature and Time Durations of Virus Inactivation on Results of Real-time Fluorescence PCR Testing of COVID-19 Viruses

Summary:The novel coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan,Hubei province of China in January 2020.This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2.Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13,2020 and throat swabs were taken.The swabs were stored at room tempcrature(20-25℃),then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses(56℃for 30,45,60 min;65,70,80℃for 10,15,20 min).Control aliquots were stored at room temperature for 60 min.Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2.Regardless of inactivation temperature and time,7 of 12 cases(58.3%)tested were positive for SARS-CoV-2 by PCR,and cycle threshold values were similar.These results suggest that virus inactivation parameters exert minimal infuence on PCR test results.Inactivation at 65℃for 10 min may be sufficient to ensure safe,reliable testing.

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.

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.

基于主成分分析的多重定量PCR荧光串扰校正

聚合酶链式反应(PCR)是分子生物学常用的检测手段,主要用于对生物的DNA或RNA进行检测。由于荧光光谱重叠和滤光片过滤带宽限制,检测时所获得的荧光数据通常会包含荧光通道之间的串扰,串扰的存在使PCR结果分析变得复杂,并可能影响最终的检测结果。选择合适的光学元件,并确定通道间的补偿矩阵,可以降低甚至消除荧光串扰。目前荧光补偿矩阵大多通过迭代计算获得,还没有一种简单的方法可以从混合的多通道荧光数据中找到荧光补偿矩阵。为了快速获得荧光补偿矩阵,减小计算量,采用主成分分析法(PCA)中确定主成分的方式,基于搭建的测试平台进行单一染料实验,获得染料的荧光信号在各个检测通道的分布情况,计算得到荧光补偿矩阵。通过分析补偿矩阵,发现对于搭建的硬件系统,Cy5染料对Cy5.5通道串扰较大,串扰比例为8.76%,同时Cy5.5染料对Cy5通道串扰影响也相对较大,比例约为6.2%;其次是ROX染料对HEX通道串扰,比例约为2.68%;HEX染料对FAM通道串扰,比例约为1.58%;FAM染料对HEX通道串扰相对较小,比例约为0.25%,其余通道无明显串扰,与荧光光谱反映的结果一致。采用得到的荧光补偿矩阵对单一染料实验得到的原始荧光数据进行处理,有效去除了非目标通道的荧光串扰,实现了荧光通道数据的解耦,验证了方法的可行性。最后设计了染料颜色分辨实验,将不同浓度的多种染料进行组合测试,并采用所提出的方法将得到的数据进行荧光补偿。实验结果表明,荧光通道各自的线性相关性较高,五个荧光通道的线性相关系数r均大于0.99,该结果进一步验证了该补偿方法的有效性。

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.

致牛腹泻4种细菌多重荧光定量PCR检测方法的建立及应用

随着规模化和集约化养牛业的不断发展,多种致病菌混合感染造成牛腹泻严重制约了养牛业的快速发展,对腹泻病原进行快速准确鉴别诊断对疫病防控至关重要。根据沙门氏菌的ivnA基因、大肠埃希氏菌的23S rRNA基因、产气荚膜梭菌的plc基因以及志贺氏菌的ipaH基因的保守区域建立了可同时检测这4种细菌的多重荧光定量PCR检测方法,并对其特异性、灵敏性和重复性进行了研究。结果显示,建立的标准曲线线性关系良好;优化后的检测方法仅可特异性扩增出4种目标细菌;对大肠埃希氏菌、沙门氏菌、志贺氏菌和产气荚膜梭菌的最低检出限分别为9.4×10~1copies/μL、1.2×10~2copies/μL、9.1×10~1copies/μL和1.4×10~3copies/μL,该方法的灵敏性高于普通单项PCR检测方法10倍;批内、批间差异均小于5%。用此方法检测人工感染病料,每份病料均检测出对应攻毒细菌的荧光信号。以上结果表明,所建立的多重荧光定量PCR方法特异性强、灵敏度高、重复性好,可用于致牛腹泻细菌的实验室检测。

基于多重实时荧光定量PCR技术检测高危型人乳头瘤病毒E6/E7 mRNA的临床研究

目的 研究基于多重实时荧光定量PCR技术检测高危型人乳头瘤病毒(HPV)E6/E7 mRNA的价值。方法 选择2022年1月至2022年7月我院进行宫颈癌筛查的患者60例,分别采取多重实时荧光定量PCR技术测定高危型人乳头瘤病毒E6/E7 mRNA情况,并将病理学检查结果作为金标准,分析基于多重实时荧光定量PCR技术检测高危型人乳头瘤病毒E6/E7 mRNA的价值。结果 HPV 16、18、31、33等九类型的最低检测为10 copies/μL,但HPV 51、52、56、58等五类型最低检测是100 copies/μL。另外通过多重实时荧光定量PCR检测高危HPV E6/E7 mRNA和生殖道内常见病原菌例如大肠埃希菌、取淋病奈瑟菌、梅毒螺旋体、金黄色葡萄球菌等,其结果均显示阴性,且检测结果的特异性良好。薄层液基细胞学的检查结果与病理学检查并无差别(P>0.05)。多重实时荧光定量PCR技术的符合率91.67%(55/60),灵敏度83.33%(15/18),特异度95.24%(40/42)。结论 基于多重实时荧光定量PCR技术在HPV E6/E7 mRNA检测中意义重大,存在较高的灵敏度及特异度,可成为宫颈病变筛查的主要方式。

4种致牛腹泻病毒多重荧光定量PCR检测方法的建立及应用

【目的】建立牛病毒性腹泻病毒(BVDV)、牛冠状病毒(BCoV)、牛轮状病毒(BRV)、牛恶性卡他热病毒(MCFV)4种致牛腹泻病毒多重荧光定量PCR检测方法,为BVDV、BCoV、BRV和MCFV的鉴别诊断提供技术支持。【方法】根据BVDV的5′UTR基因、BCoV的N基因、BRV的VP6基因和MCFV的ORF9基因的保守区域设计引物,克隆目的基因构建标准阳性重组质粒,建立可同时检测4种致牛腹泻病毒的多重荧光定量PCR检测方法,并对该方法的特异性、敏感性和重复性进行评价。用建立的多重荧光定量PCR检测方法对76份临床样品进行检测,并与常规PCR检测方法的结果进行比较。【结果】建立了BVDV、BCoV、BRV和MCFV多重荧光定量PCR检测方法,该方法建立的标准曲线线性关系良好,仅可特异性扩增出4种目标基因片段;对BVDV、MCFV、BRV和BCoV的最低检出限分别为9.4×10^(1),1.4×10^(3),9.1×10^(1)和1.2×10^(2)拷贝/μL,与普通PCR相比,灵敏性高出10~1000倍;批内、批间差异均小于5%。76份临床样品检测结果显示,多重荧光定量PCR检测结果与普通PCR检测结果的符合率分别为98.7%(BCoV)、97.4%(BRV)、100.0%(BVDV)和100.0%(MCFV)。【结论】建立了BVDV、BCoV、BRV和MCFV多重荧光定量PCR检测方法,该方法特异性强、灵敏度高、重复性好,可用于牛腹泻病毒病原的实验室检测。

Effects of Wei Chang An on expression of multiple genes in human gastric cancer grafted onto nude mice

AIM： To investigate the expression of multiple genes in Chinese jianpi herbal recipe Wei Chang An （WCA） in human gastric cancer cell line SGC-7901. METHODS： A human gastric adenocarcinoma cell line SGC-7902 grafted onto nude mice was used as the animal model. The mice were randomly divided into 3 groups, one control and the two representing experimental conditions. Animals in the two experimental groups received either WCA over a 34-d period or 5-fluorouracil （5-FU） over 6-d period starting at 8th d after grafting. Control animals received saline on an identical schedule. Animals were killed 41 d after being grafted. The expression profiles in paired WCA treated gastric cancer samples and the N.S. control samples were studied by using a cDNA array representing 14181 cDNA clusters. The alterations in gene expression levels were confirmed by Real-time Quantitative polymerase chain reaction （qPCR）. RESULTS： When compared with controls, the average tumor inhibitory rate in WCA group was 44.32% ± 5.67% and 5-FU 47.04% ± 22.33% （P 〈 0.01, respectively）. The average labeling index （LI） for PCNA in WCA group and 5-FU group was significantly decreased compared with the control group. Apoptotic index （AI） was significantly increased to 9.72% ± 4.52% using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling （TUNEL） method in WCA group compared with the controls 2.45% ± 2.37%. 5-FU group was also found to have a significantly increased AI compared with the controls. The expression of cleaved Caspase-3 in WCA group and 5-FU group was significantly increased compared with the control group respectively. There were 45 different expressed sequence tags （ESTs） among the control sample pool and WCA sample pool. There were 24 ESTs up-regulated in WCA samples and 21 ESTs down-regulated. By using qPCR, the expression level of Stat3, rap2 interacting protein x （RIPX）, regulator of differentiation 1 （ROD1） and Bcl-2 was lower in WCA group than that in control group respectively. By using SP immunohistochemical method the expression of Phospho-Stat3 （Tyr705） and Bcl-2 in WCA group and 5-FU group was significantly decreased compared with the control group respectively. CONCLUSION： WCA could inhibit gastric cancer cell SGC-7901 growth in vivo. WCA could induce gastric cancer cell apoptosis and suppress proliferation. Its mechanisms might be involved in the down-regulation of Star3, RIPX, ROD1 and Bcl-2 gene.

牛呼吸疾病综合征七病原联合检测多重qPCR方法的建立

为了提高牛呼吸疾病综合征多病原混合感染的临床诊断效率,以牛支原体(Mycoplasma bovis,M.b)oppD/F基因、多杀性巴氏杆菌(Pasteurella multocida,P.m)ompH基因、溶血性曼氏杆菌(Mannheimia haemolytica,M.h)gcp基因、牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)gB基因、牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)以及牛副流感病毒(bovine parainfluenza virus type,BPIV) 3型a和c基因型(BPIV-3a,-3c)的N基因等为检测靶标,分别设计特异性引物和Taqman探针,通过优化反应条件,采用3管7联的组合方式建立了7种病原体的多联实时荧光定量PCR检测方法。结果显示,该方法仅对本试验的7种病原有特异性反应,与其他常见病原无交叉反应。对M.b、P.m、M.h、IBRV、BRSV、BPIV-3a和BPIV-3c质粒标准品的最低检测限分别为102、102、101、102、102、102和101拷贝/μL。组内变异系数小于2.5%,组间变异系数小于5.5%。平行应用该方法和常规PCR方法对临床采集的115份有呼吸道症状牛的鼻拭子进行检测,P.m阳性率36.65%,M.b阳性率27.83%,M.h阳性率25.22%,IBRV阳性率11.30%,BPIV-3c阳性率8.57%,BRSV阳性率0.95%;其中混合感染率为26.1%。共检测到11种混合感染模式,主要由M.b与其他病原体的混合感染,占72.7%(8/11);M.b/P.m混合感染的检出率最高,占60%(18/30);M.b、P.m、M.h在混合感染中出现率排前三,其占比分别为73.3%(22/30)、73.3%(22/30)和43.3%(13/30);其次为IBRV,占26.7%(8/30);BPIV-3c占13.3%(4/30)。以上结果表明,该方法具有较高的分析敏感性和特异性,可用于牛呼吸疾病综合征多病原感染的联合检测。

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.

Altered gene expression reveals molecular mechanisms underlying oridonin-induced apoptosis of multiple myeloma LP-1 cells

Objective： To investigate the effect of oridonin on proliferation and invasion of human multiple myeloma LP-1 ceils and the underlying mechanism. Methods： LP-1 cells in culture medium in vitro were treated with oridonin at the different concentration Cell proliferation was measured by Microwave Theory and Techniques （MTT） assay and cell apoptotic rate was detected by flow cytometry. Morphology of cell apoptosis was observed by transmission electron microscope. Expressions of Bax, Bcl-2, Caspase-3, NFqcB as well as I-~B mRNA were detected by real-time PCR. Results： The MTT assays and flow cytometry revealed that oridonin could inhibit the growth of LP-1 cells and cause apoptosis significantly; the suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis were found under a transmission electron microscope after the cells were treated with oridonin at 25 ~rnol/L for 24 h. Along with the apoptotic process, Bcl-2, Caspase-3,NF-r,.B gene expressions were down-regulated （P〈0.05）. On the contrast, the Bax and I-~zB gene expressions were up-regulated （P〈0.05）. Conclusion： Oridonin could inhibit the proliferation of LP-1 cells via inducing apoptosis. We concluded that oridonin induces apoptosis in LP-1 cells via activation of caspase-3 as well as down-regulation of Bcl-2 and up-regulation of Bax expression. The results suggested that oridonin could induce apoptosis of LP-1 cells through mitochondria- and caspase3-dependent pathways. Meanwhile, the inhibition of NF-r,_B and the activation of I-~B indicate pro-apoptotic stimuli. In one word, oridonin might be an important potential anti-myeloma reagent.

多重荧光PCR方法与血清凝集方法对沙门氏菌分型鉴定的结果对比

目的 探讨多重荧光PCR方法和血清凝集方法在沙门氏菌分型鉴定结果中的意义。方法 选取扬州市邗江区疾病预防控制中心在2022年9月—2023年6月接诊的4940例进行门诊健康证筛查的患者,上述患者均接受血清凝集方法以及多重荧光PCR方法进行检测,探究两种检测方式在沙门氏菌中的检出率以及沙门氏菌分型鉴定结果。结果 多重荧光PCR方法检出效能与血清凝集方法检出效能间无统计学意义(P>0.05);在沙门氏菌分型鉴定结果中对比,多重荧光PCR鉴定方法所检测出的沙门氏菌分型中,肠炎沙门氏菌(1株),鼠伤寒沙门氏菌(1株),B群沙门氏菌(3株)较血清凝集鉴定方法检出率高,但组间对无统计学意义(P>0.05)。结论 多重荧光PCR方法和血清凝集方法在沙门氏菌检测和分型鉴定中各有优势,在特定的应用场景下可以选择适合的方法来提高检测效率和准确性,但本研究存在一定的局限性、年限短、样本量少,今后可加大样本量进一步研究。