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 samples. 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.

Synchronous Detection of DNA/RNA of Four Shrimp Viruses by Real-time Fluorescence Quantitative RT-PCR

[Objective]This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR,to improve the efficiency of inspection and quarantine. [Method]A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA /RNA of four shrimp viruses(WSSV,IHHNV,TSV and YHV). [Result]The optimized real-time fluorescence quantitative RT-PCR system generated typical amplification curves with high amplification efficiencies(E = 1. 06,1. 07,0. 92 and 0. 92,respectively),good linear relationship(r = 1),uniform repeatability(standard deviation = 0. 05- 0. 46; variation coefficient = 0. 26%- 1. 62%) and high sensitivity,exhibiting no significant differences compared with real-time fluorescence quantitative PCR(average error of Ct value = 0. 04- 0. 40; T = 0. 53- 2. 50; P > 0. 05). The total detection time was about 1 h. [Conclusion]The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV,IHHNV,TSV and YHV.

Detection and clinical significance of multidrug resistance-1 mRNA in bone marrow cells in children with acute lymphoblastic leukemia by real-time fluorescence quantitative RT-PCR

Objective: Multidrug resistance(MDR) is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia(ALL) which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence- quantitative reverse transcription polymerase-chain reaction(FQ-RT-PCR), and combine minimal residual desease(MRD) detection by flow cytometry(FCM) and to study their relationship with treatment response and prognosis of ALL. Methods:The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission(CR), 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9～ 101 months, with a median of 64 months. Results:Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group(P > 0.05), but significantly higher in the recurrence group than that in newly diagnosed disease group and control group(0.50±0.55 vs. 0.09±0.26 and 0.12±0.23, P < 0.05). 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3～ 6 years and over 6 years duration(0.63 ± 0.56 vs. 0.11 ± 0.12 and 0.04 ± 0.06, P < 0.01). For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC > 50×109 group and WBC<50×109 group(P > 0.05). In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD>103 group than that in MRD<103 group(0.39±0.47 vs. 0.03± 0.03, P < 0.05). Conclusion:The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrow cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration(within 3 years). Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.

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 pathogenesis,early clinical diagnosis 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 16 S rRNA gene sequences of common Lactobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template,and L. acidophilus in fermented material was conducted the quantitative determination by real-time quantitative PCR( RT-PCR). Analysis on RT-PCR results shown that contents of L. acidophilus 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 applied to the detection of L. acidophilus in fermented material.

Real-time fluorescent quantitative immuno-PCR method for determination of fluoranthene in water samples with a molecular beacon

A reliable and sensitive competitive real-time fluorescent quantitative immuno-PCR (RTFQ-IPCR) assay using a molecular beacon was developed for the determination of trace fluoranthene (FL) in the environment.Under optimized assay conditions,FL can be determined in the concentration range from 1 fg/mL to 100 ng/mL,with y=0.194x + 7.859,and a correlation coefficient of 0.967 was identified,with a detection limit of 0.6 fg/mL.Environmental water samples were successfully analyzed,recovery was between 90% and 116%,with intra-day relative standard deviation (RSD) of 6.7%-12.8% and inter-day RSD of 8.4%-15.2%.The results obtained from RTFQ-IPCR were confirmed by ELISA,showing good accuracy and suitability to analyze FL in field samples.As a highly sensitive method,the molecular beacon-based RTFQ-IPCR is acceptable and promising for providing reliable test results to make environmental decisions.

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

Real-time 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 disadvantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the application and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.

TaqMan real-time fluorescent quantitative RT-PCR in detection of macrophage inflammatory protein-2γ mRNA in myocarditis murine

Objective: To study the role of macrophage inflammatory protein (MIP)-2γ in myocarditis pathogenesis in BALB/c mice. Methods: The relationship between the progression of Coxsarckie virus B3(CVB3) viral myocarditis and experimental autoimmune myocarditis and MIP-2γ mRNA expression in mouse was studied by TaqMan real-time fluorescent quantitative RT-PCR. Results: MIP-2γ mRNA expression rose on 3 to 5 d after CVB3 infection, reached peak on 7 d, and returned to normal level until 14 d, which corresponded well with the disease course. The MIP-2γ mRNA expression level rose significantly on the day 18 d after immunization with porcine cardiac myosin, which was consistent with pathological examination. Conclusion: MIP-2γ may be involved in the pathogenesis of myocarditis.

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 replications.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 4;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 organisms and products in China.

Real-Time PCR探针法定量检测沙门菌方法的建立及应用

目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。

Real-time TaqMan RT-PCR快速检测犬瘟热病毒方法的研究

按照犬瘟热(CDV)N基因序列,设计合成了特异性引物和探针,经各反应条件的优化,建立了Real-time荧光定量RT-PCR技术,对细胞培养物、肝脏、肺脏、脑、脾脏、淋巴结以及鼻腔拭子等组织病料中的CDV进行了特异性检测和敏感性试验。同时,利用建立的Real-time荧光定量RT-PCR方法与常规RT-PCR以及韩国BIOINDIST生产的BIT RAPID CDV检测试剂盒对57份临床样品进行了检测。结果:用20pmol/mL的引物浓度各1uL和20pmol/mL的探针浓度0．3uL,获得的荧光信号最强,曲线平滑。敏感性高,可检测到1．24×10—3ng/uL的病毒RNA;特异性强,与NDV、AIV、NiPV等RNA病毒不发生交叉反应。试验重复性的变异系数(CV)分别为2．3%、2．5%和4．2%;与常规RT-PCR和BIOINDIST生产的BIT RAPID CDV检测试剂盒相比较,该方法具有快速、特异、敏感、可定量,并可同时检测大量样品等优点。

3种猪繁殖障碍性病毒Real-time PCR快速检测方法的建立

【目的】建立可同时检测猪伪狂犬病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒Ⅱ型(PCV2)的多重实时荧光定量PCR方法。【方法】根据GenBank数据库中PRV、PPV和PCV2的核苷酸序列,设计3对特异性引物和探针,以10倍系列稀释的阳性质粒为模板,优化反应条件,建立检测PRV、PPV和PCV2的多重Real-time PCR方法,并对其敏感性、重复性和特异性进行检验;分别采用单项和多重Real-time PCR方法,对临床收集的42份疑似病料进行检测,比较2种方法的符合率。【结果】特异性和灵敏度试验表明,建立的多重Real-time PCR检测方法具有高度特异性,与其他病原无明显交叉反应;检测灵敏度高,可检出1.0×101拷贝/μL的阳性质粒或1TCID50/mL的病毒样品。用多重Real-time PCR对42份临床疑似病料进行检测,其检测结果与单重Real-time PCR结果完全一致,表明多重Real-time PCR方法是可行的。【结论】建立了可同时检测PRV、PPV和PCV2的多重Real-time PCR方法,该法具有快速、灵敏、特异和重复性好等优点。

鸽微RNA病毒实时荧光定量RT-PCR检测方法的建立

旨在建立鸽微RNA病毒(pigeon megrivirus,PiMeV)实时荧光定量RT-PCR检测方法。本研究根据GenBank中PiMeVs序列特征设计特异性检测引物,从信鸽粪便中检测到PiMeV阳性(命名为PiMeV-CHN001株),并对其3 C基因进行核苷酸同源性比较和遗传进化分析,明确其基因特征后,设计特异性实时荧光定量RT-PCR检测(RT-qPCR)引物组,建立检测PiMeV的RT-qPCR方法。结果显示:PiMeV-CHN001株3 C基因全长为591 bp,编码197个氨基酸,和其他2株野鸽源PiMeV(MeV-B1株和MeV-B2株)核苷酸相似性分别为89.5%和92.0%。建立的检测PiMeV的RT-qPCR方法的标准曲线Y轴截距为37.93,斜率为-3.335,相关系数为1.00,扩增效率为99.4%。特异性强,仅PiMeV出现特异性扩增信号和特异性峰值[Tm值为(81.69±0.22)℃],对鸽源禽流感病毒(avian influenza virus,AIV)、鸽源禽I型副黏病毒(pigeon paramyxovirus type I,PPMV-1)、鸽输血传播病毒(pigeon torque teno virus,PTTV)、鸽腺病毒(pigeon adenovirus,PiAd)及鸽圆环病毒(pigeon circovirus,PiCV)检测均未见特异性扩增信号;敏感性优,最低检测限为54.0拷贝·μL^(-1);重复性好,批内和批间变异系数均低于1.5%。用建立的检测方法对42份信鸽粪便样品进行检测,发现2份阳性样品(阳性率为4.76%)。本研究首次证实我国大陆地区信鸽中存在PiMeV,丰富了PiMeV宿主谱信息;建立的RT-qPCR方法为后续开展PiMeV流行病学研究提供支撑。

美洲鳗鲡腺瘤病毒(AEAdoV)普通PCR和SYBR Green I实时荧光定量PCR检测方法的建立及应用

为建立美洲鳗鲡腺瘤病毒(AEAdoV)的检测方法,根据AEAdoV福建株(AEAdoVFJ)的superfamily 3 helicases(S3H)序列,设计引物,建立了AEAdoV的普通PCR和qPCR检测方法;进一步评价检测方法的灵敏性、特异性及重复性,利用2种方法对美洲鳗鲡“出血性烂鳃”病料进行了检测,并对美洲鳗鲡体内不同组织的病毒含量进行分析。结果显示,普通PCR扩增的目的片段长度约300 bp,利用其构建的qPCR质粒标准品,其拷贝数与qPCR阈值循环数(C_(t))线性关系良好,线性范围广,标准曲线相关系数(R2)达到0.999,扩增效率为105.067%。建立的普通PCR法和qPCR的最低检测AEAdoV拷贝数分别为100个和10个。2种方法均可特异性检测AEAdoV,而对蛙虹彩病毒(RGV)、鳗鲡疱疹病毒(AngHV)、鲤疱疹病毒(KHV)、对虾白斑综合征病毒(WSSV)、日本鳗鲡内皮细胞坏死病毒(JEAdoV)和花鳗鲡腺瘤病毒(MEAdoV)均无扩增反应。qPCR法的组内和组间变异系数均小于2%,表明其重复性良好。临床应用结果显示,35份美洲鳗鲡“出血性烂鳃”病料,采用普通PCR法的AEAdoV检出率为82.8%,而qPCR法的AEAdoV检出率为97%。对美洲鳗鲡不同组织的病毒含量分析结果显示,心脏、肝脏、鳃、鳍的AEAdoV相对含量较高,而黏液、皮肤和脾脏的病毒含量相对较低。研究表明,建立的AEAdoV的灵敏度高、特异性强的普通PCR和qPCR检测方法,证实AEAdoV与美洲鳗鲡“出血性烂鳃”病密切相关,且在感染鳗鲡主要组织中都存在。实验结果对于研究AEAdoV的致病性,开展其流行情况和病原学情况分析具有重要意义。

基于LNA-TaqMan探针实时荧光定量PCR检测技术的药用黄精掺伪研究

目的:建立一种快速、灵敏、有效的实时荧光定量聚合酶链式反应(PCR)方法,用于检测湖北黄精掺伪药用黄精。方法:基于锁核酸(LNA)-TaqMan探针实时荧光定量PCR技术,利用不同基原药用黄精样品的叶绿体DNA中trnC-petN基因序列差异,根据常见混伪品湖北黄精特异性差异位点设计筛选探针引物,并对引物及LNA-TaqMan探针的特异性进行验证。根据扩增曲线临界循环数(Ct)值的差值计算湖北黄精掺伪比例。结果:基于LNA-TaqMan探针能够特异性地检测出湖北黄精并确定掺伪比例,在湖北黄精掺伪1%时,仍可稳定检出。结论:该方法简便准确、稳定可靠,可以用于药用黄精掺伪湖北黄精定量检测。

牛呼吸道合胞体病毒一步法实时荧光定量PCR检测方法的建立及应用

为建立一种牛呼吸道合胞体病毒(Bovine respiratory syncytial virus, BRSV)快速简便的检测方法,本研究基于BRSV M基因保守区序列,设计特异性引物及探针,通过优化反应条件,建立用于BRSV检测的一步法实时荧光定量PCR,并验证了该方法的敏感性、特异性和重复性;同时利用建立的检测方法对采集的临床样本进行检测。结果表明:本研究所建立的BRSV荧光定量检测方法,其特异性好,仅对BRSV存在特异性扩增;敏感性高,最低可达10 copies/μL;稳定性好,组内变异系数和组间变异系数小。使用所建立的BRSV一步法实时荧光定量PCR对宁夏地区94份临床样品进行检测,阳性率为5.3%(5/94)。上述结果表明,本研究建立的检测方法可为BRSV的快速诊断提供有力的技术支持。

Real-time PCR方法检测高脂饲喂小鼠肝脏CYP2A5的表达

为了建立非酒精性脂肪肝(NAFLD)模型小鼠肝组织中CYP2A5表达的实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)方法。高脂饮食诱导小鼠至8周,采用qRT-PCR方法检测小鼠肝脏中CYP2A5的表达水平,同时评价该方法的特异性。建立了小鼠肝组织中CYP2A5的SYBR Green实时荧光定量PCR检测方法,结果显示,该方法的溶解曲线为单峰,同时核酸电泳显示一条特异性条带。检测结果表明,高脂诱导的NAFLD小鼠肝组织中CYP2A5的表达水平极明显高于正常对照组(P<0.01)。表明该实时荧光定量PCR方法,特异性好、灵敏度强,为进一步研究小鼠CYP2A5的表达提供了试验基础。

Real-time PCR检测大豆CONSTANS基因在不同光照条件下的表达

采用实时荧光定量PCR方法检测了不同感光大豆品种的CO(CONSTANS)基因在不同光照条件下的mRNA水平变化。结果表明:早熟品种东农49和晚熟品种东农42各自在短日照(8h/16h光/暗)下会比长日照(16 h/8 h光/暗)下表达有所增强,同时东农49中CO基因的表达要高于东农42,它们的表达峰值均出现在光暗交界处。东农42由长日照或短日照移入完全光或完全暗条件时,暗下CO基因的表达量剧增,并且大致维持之前长日照和短日照的表达规律。CO基因的表达有很强的昼夜节律性,暗下利于其表达,并且在不同感光品种中表达有所不同。

禽源大肠杆菌荧光定量PCR方法的构建及应用

为建立一种灵敏、准确且快速检测禽源大肠杆菌的方法,根据禽源大肠杆菌uidA基因的一节特异性保守序列设计引物,建立用于禽源大肠杆菌的荧光定量PCR(qPCR)检验方法,并对其各方面进行评价。试验数据表明所建立的qPCR方法的Ct值与标准品在4.69×10^(2)~4.69×10^(9 )copies/μL范围内存在优良的线性关系,线性相关系数为R2=0.993;该方法标准曲线方程为y=-3.2786x+39.032,熔解曲线表现为单峰,不存在非特异性扩增。对重组质粒标准品的最低检测浓度为4.69×10^(2) copies/μL,是普通PCR方法的1000倍。该方法检测临床样本阳性率为68.3%(41/60),普通PCR阳性检出率为23.3%(14/60),阳性检出率高出45%。此次研究建立的检测禽源大肠杆菌的荧光定量PCR检测方法可用于禽源大肠杆菌的快速诊断,对于禽大肠杆菌病的监测具有重要意义。

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.