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 gener- ated typical amplification curves with high amplification efficiencies （E = 1.06, 1.07, 0.92 and 0.92, respectively）, good hnear 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 re- al-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 × 10^9 group（P〉 0.05）. In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD〉10a group than that in MRD〈10a 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 marrowy 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.

THE DETECTION OF MDR1 GENE EXPRESSION USING FLUOROGENIC PROBE QUANTITATIVE RT-PCR METHOD

Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cancer. Methods: The fluorogenic quantitative RT-PCR method for detection of the expression of MDR1 gene was established. K562/ADM and K562 cell lines or 45 tumor tissues from patients with lung cancer were examined on PE Applied Biosystems 7700 Sequence Detection machine. Results: the average levels of MDR1 gene expression in K562/ADM cells and K562 cells were (6.86±0.65)× 107 copies/μg RNA and (8.49±0.67)×105 copies/μg RNA, respectively. The former was 80.8 times greater than the latter. Each sample was measured 10 times and the coefficient variation (CV) was 9.5% and 7.9%, respectively. Various levels of MDR1 gene expression were detected in 12 of 45 patients with lung cancer. Conclusion: Quantitative detection of MDR1 gene expression in tumor cells was achieved by using FQ-RT-PCR. FQ-RT-PCR is an accurate, and sensitive method and easy to perform. Using this method, low levels of MDR1 gene expression could be detected in 24% of the patients with lung cancer.

Establishment of a Real-time Fluorescent Quantitative RTPCR Method for Detecting NP Gene of Class Ⅰ Newcastle Disease Virus(NDV)

Newcastle disease( ND) is one of the most serious infectious diseases that infect the poultry industry.There is only one serotype of Newcastle disease virus( NDV),but NDVs can be divided into two distinct classes( class Ⅰ,and class Ⅱ) according to their genetic relationship.To develop a method for rapid quantitative detection of class Ⅰ NDV,a pair of primers and a TaqM an probe were designed and synthesized according to the conservative sequence of NP gene of class Ⅰ NDV.The positive recombinant plasmid harboring NP gene of JS-18-05 isolate was used as a positive template to establish the standard curve.A real-time fluorescent quantitative RT-PCR method was established for rapid detection of class Ⅰ NDV with strong specificity,high sensitivity and good repeatability.The established method exhibited a good linear relationship within the concentration of 102 to 108 copies of NDV,by which 1 μl of 10 copy of NDV nucleic acid could be detected in the initial template.Compared with conventional virus isolation methods,the established method had similar sensitivity and led to the same results in detecting33 class Ⅰ,class Ⅱ NDV isolates.The study provided the basis for rapid quantitative detection of class Ⅰ NDVs and further clarification of their pathogenicity and pathogenic mechanism in poultry.

基于定量CT身体组分、^(18)F-FDG PET/CT显像预测手术联合新辅助化疗治疗早期乳腺癌预后的价值

目的 分析定量CT (QCT)身体组分、^(18)F-氟脱氧葡萄糖正电子发射型计算机断层显像(^(18)F-FDG PET-CT)对手术联合新辅助化疗(NAC)治疗早期乳腺癌患者预后的预测效能,为临床治疗提供参考。方法 选取2020年3月至2022年10月于南阳市第二人民医院接受手术联合NAC治疗的82例早期乳腺癌患者纳入研究,在NAC前和化疗1、3个周期后检测记录QCT参数[L_(1)、L_(2)水平的皮下脂肪面积(SFA)、内脏脂肪面积(VFA)、骨密度(BMD)、L_(3)水平的椎旁肌肉面积(TMA)]、^(18)F-FDG PET/CT显像的最大标准摄取值(SUV_(max))、肿瘤代谢体积(MTV)。NAC结束后进行手术,随访12个月(失访2例),依据有无复发转移分为预后良好组43例和预后不良组37例,比较不同预后患者的QCT参数、^(18)F-FDG PET/CT显像指标,采用受试者工作特征(ROC)曲线获取曲线下面积(AUC)分析其对早期乳腺癌患者预后的预测效能。结果化疗1个周期后,预后良好组患者的SFA、BMD、VFA、MA水平分别为(45.23±4.07) cm^(2)、(128.97±26.53) mg/m^(2)、(78.07±6.69) cm^(2)、(37.36±5.74) cm^(2),明显高于预后不良组的(42.52±3.32) cm^(2)、(112.54±25.82) mg/m^(2)、(73.73±7.25) cm^(2)、(32.94±5.31) cm^(2),差异均有统计学意义(P<0.05);化疗3个周期后,预后良好组患者的SFA、BMD、VFA、MA水平分别为(40.95±3.92) cm^(2)、(113.55±15.87) mg/m^(2)、(73.59±6.17) cm^(2)、(32.67±4.98) cm^(2),明显高于预后不良组的(37.51±3.56) cm^(2)、(95.18±17.45) mg/m^(2)、(70.30±5.14) cm^(2)、(28.52±4.42) cm^(2),差异均有统计学意义(P<0.05);化疗1个周期后,预后良好组患者的SUV_(max)、MTV分别为5.43±1.25、(3.86±0.87)×10^(4)mm,明显低于预后不良组的6.04±1.07、(4.27±0.85)×10^(4)mm,差异均有统计学意义(P<0.05);化疗3个周期后,预后良好组患者的SUV_(max)、MTV分别为3.94±1.06、(2.61±0.70)×10^(4)mm,明显低于预后不良组的4.73±1.21、(3.05±0.93)×10^(4)mm,差异均有统计学意义(P<0.05);经ROC曲线分析结果显示,SFA、BMD、VFA、MA、SUV_(max)、MTV联合预测手术联合NAC治疗早期乳腺癌患者预后的AUC分别为0.898 (95%CI:0.809~0.954)、0.919 (95%CI:0.836~0.968)。结论 NAC过程中检测QCT参数、^(18)F-FDG PET/CT显像指标可预测手术联合NAC治疗早期乳腺癌患者的预后,联合预测的价值较高。

量化积分绩效分配模式在消毒供应中心工勤人员中的应用研究

目的探讨量化积分在消毒供应中心工勤人员绩效分配中的应用效果,为消毒供应中心提供一种有效的绩效分配方法。方法采用前-后对照研究方法,选取2020年1月至12月本院消毒供应中心绩效分配模式为对照组,选取2021年1月至12月本院消毒供应中心绩效分配模式为试验组。对照组采用按工勤人员工龄绩效分配模式,试验组采用量化积分绩效分配模式,即从技术层级、工作质量、工作效率、工作量方面实施量化绩效分配模式。比较改进前后两组工勤人员工作效率、工作质量缺陷发生率(包装器械缺失、器械种类错误、包装标识错误、收送物品错误、信息系统操作错误)、临床科室满意度情况。结果实施量化积分绩效分配模式后,试验组工勤人员工作效率和临床科室满意度均高于对照组,工作缺陷发生率低于对照组,两组比较,差异具有统计学意义(均P<0.05)。结论量化积分绩效分配模式在消毒供应中心工勤人员中的应用,能够提高工勤人员的工作能动性,提高工作效率和工作质量。

定量RT-PCR及其在植物学研究中的应用

定量RT-PCR(Quantitative reverse transcriptase-PCR)是在反转录和定量PCR的基础上发展起来的一种特异性检测基因表达的技术。主要包括相对定量RT-PCR(Relative quantitative RT-PCR)、竞争性定量RT-PCR(Competitive quan-titative RT-PCR)、比较定量RT-PCR(Comparative quantitative RT-PCR)和实时定量RT-PCR(Real time quantitative RT-PCR)四种。目前定量RT-PCR在植物学研究中的应用越来越广泛,如植物营养学研究、植物发育学研究、植物抗逆机理研究、转基因植物的检测、病原菌的检测、植物与微生物互作机理研究、植物抗病性检测等方面。本文综述了定量RT-PCR的原理及在植物学中的应用。

Establishment and Application of Digital RT-PCR Assay for Detection of Avian Influenza Virus H9 Subtype

A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus.

The application of sequence specific primer and RT-PCR to LRRK2 gene polymorphism typing

Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA markers with known genotypes by use of quantitative fluorescence real-time PCR(RT-PCR).100 cases of PD samples with unknown genotypes were tested,and verified by use of polymerase chain reaction linked restriction fragment length polymorphism(PCR-RLFP).Results:The genotyping results of DNA markers proved to be correct,and 100 cases of samples to be tested had a completely consistent genotyping result with PCR-RLFP genotyping result.Conclusions:Sequence specific primer and quantitative fluorescence RT-PCR can successfully make a genotyping for disease susceptibility loci R1628P and G2385R of LRRK2.

综合交通各种运输方式技术经济特征与比较优势研究

为支撑国家综合立体交通网规划中各种运输方式合理布局,研究了各种运输方式的技术经济特征与比较优势水平。首先,聚焦成本和效用分析,研究提出了运输方式技术经济特征与比较优势评价指标体系;然后,从费用化比较的角度,甄选指标体系中的代表性指标,构建指标量化模型,提出了指标计算方法。并以长江通道的上海—重庆为例,测算了各种运输方式的经济费用、时间价值、安全成本和绿色费用。测算结果表明了各种运输方式在经济性、时效性(便捷性)、安全性、绿色性等方面的技术经济特征与比较优势量化水平,如在绿色性方面,最具优势的运输方式是管道(货运),其次是铁路、水运、公路,航空的绿色治理费用最高。

国内外工作旺盛感的研究计量可视化与比较分析

为全面把握国内外工作旺盛感的研究热点及趋势变化。基于中国知网(2013—2022)与Web of Science(2005—2022)核心数据库相关文献,运用Citespace软件,通过对国内外该领域的学者发文量、核心作者合作、关键词共现、聚类、研究前沿及知识基础等方面进行比较分析。结果发现:在发文量上,2018年后国内外均呈上升趋势;在核心作者合作上,国内外形成小规模的团队,但联系较弱,应加强不同团队间跨域合作;在研究热点和前沿上,国内外具有趋同性,即国内外学者均持续关注旺盛感的前因与积极结果,而差异性在国内学者更关注多元领导风格的影响;在知识基础上,研究内容较为相似。未来国内学者可以从内容的多元化、情境的本土化、视角的层次化及方法的差异化四个方面继续深入探究。

基于3D技术的轮胎痕迹检验研究

目的研究利用轮胎立体痕迹的三维模型进行定量化、自动化比较检验的可行性;方法先利用打样膏制作轮胎压印痕迹,而后利用德国GOM三维量测系统采集轮胎压印痕迹的三维模型数据,并借助德国GOM公司的ATOS Inspect Suite 2020软件对三维模型数据进行重叠对齐比较检验;结果来自轮胎相同部位压印痕迹的三维模型重叠对齐效果好,误差小,能够反映造痕体的同一性。来自轮胎不同部位压印痕迹的三维模型重叠对齐效果差,细节特征出现多处分离,差异大,能够反映不同造痕体的差异性;结论利用德国GOM三维量测系统对轮胎压印痕迹进行定量化比较检验可行,且可实现自动化比较检验。

多溴联苯醚生物富集因子的CoMFA模型

研究19种多溴联苯醚(PBDEs)在淡水生物正颤蚓体内生物富集因子(lg B_(CF))的三维-定量构效关系(3D-QSAR).基于比较分子力场分析(CoMFA)建立训练集的3D-QSAR模型,不仅表现出显著的统计质量,而且还表现出令人满意的预测能力,具有高相关系数值(R^(2)=0.80)和交叉验证系数值(Q^(2)=0.32).该模型中立体场、静电场贡献率依次为45.1%、54.9%.基于CoMFA等高线图,多溴联苯醚的lg B_(CF)主要取决于溴原子在苯环上的位置,其次才与溴原子数正相关.

环保产业园创新创业绩效比较研究

为帮助环保产业园认识自身发展水平,基于中国环保产业园创新创业绩效评价指标体系,文章比较分析了4个案例园区的创新创业绩效。通过分析案例园区创新资源优势形成顺序,提出了分阶段的发展建议。在环保产业园起步阶段顶层设计和政府支持最为关键,其后应提升研发投入和营造良好的人才环境以推动园区产业提质,产业规模成熟的园区则应完善配套服务、信息渠道和金融支撑,为高端化发展建立保障。

民族地区乡村旅游产业进化“殊途”?——基于定性比较分析法的组态路径分析

乡村旅游产业进化对民族地区乡村振兴和实现共同富裕具有重要价值。文章借鉴经济进化论和现代资源观理论,通过自上而下的理论演绎构建乡村旅游产业进化过程模式;采用文本分析法与定性比较分析法,分析影响民族地区乡村旅游产业进化的资源要素,探索产业进化组态路径。基于广西龙脊梯田景区61家旅游企业研究发现:(1)在多种组织资源持续交互作用下,乡村旅游产业呈现“自由演化-市场选择-实现进化”的螺旋式进化趋势;(2)组织学习能力、人力资本、技术创新、知识联盟、正式制度、创业环境6要素构成5条组态路径,推动民族地区乡村旅游产业进化;(3)组织学习能力是所有组态路径的核心要素,知识联盟对初始资源劣势型乡村旅游小企业不可或缺,合理配置资源是进化的关键。研究结论揭示了民族地区乡村旅游产业进化的复杂因果本质,具有积极的实践借鉴和理论价值。

Detecting PML-RARα transcript in acute promyelocytic leukemia using real-time quantitative RT-PCR

Background Real-time quantitative RT-PCR （RQ-PCR） assay has become a vital tool to monitor residual disease of leukemia, However, the complexity and standardization of RQ-PCR should never be overlooked and the results should be interpreted cautiously in clinical conditions. We aimed to assess the methodology of RQ-PCR and its clinical applications in monitoring molecular kinetics of 36 newly diagnosed cases of acute promyelocytic leukemia patients with t （15;17） from October 2004 to December 2005.Methods All the TaqMan probe-based RQ-PCR reactions and analysis were performed on an ABI-PRISM 7500 platform, The quantitation of PML-RARα transcripts was represented by the normalized quotient, that is, PML-RARα transcript copies divided by ABL transcript copies, According to induction therapy, the patients were classed into two groups： group 1 （n=23）, three-drug combination including arsenics, all-trans retinoic acid and mitoxantrone; and group 2 （n=13）.two-drug combination from all-trans retinoic acid, arsenics and mitoxantrone.Results The sensitivity of RQ-PCR was 1 per 105 cells and 5 copies of the PML-RARα transcript could be reproducibly detected, No false positive results occurred in 40 non-acute promyelocytic leukemia samples, Optimal amplification efficiency could be attained, which was determined by the slope of the standard curves （slope： -3.2 -- -3.7）. The inter-assay and intra-assay variation coefficients of the method were 1.01% and 0.56% respectively. Although the time to attain hematological complete remission was similar in both groups, the time to achieve molecular remission of group 1 was significantly shorter than that of group 2 （61 days vs 75 days, P=0.034）. The rate of molecular remission within 70 days was higher in group 1 than in group 2 （75.00% vs 38.46%, P=0.036）, Compared with pretreatment, median reduction of the PML-RARα transcript before first consolidation therapy differed significantly between group 1 and group 2 （log scale, 3.15 vs 2.31, P=0.024）, Interestingly, we found that PML-RARα transcript levels temporarily increased in bone marrow （7 patients） and peripheral blood （22 patients） samples of patients during induction therapy in both groups.Conclusions The RQ-PCR assay is reliable for the detection of PML-RARα transcripts. Arsenics, all-trans retinoic acid and mitoxantrone triad induction treatment of acute promyelocytic leukemia is superior to two-drug combination induction therapy in terms of the molecular response.

取代嘧啶衍生物抗癌活性的CoMFA研究与分子设计

基于比较分子力场分析(CoMFA)方法研究21种取代嘧啶衍生物对乳腺癌抑制活性(S_M)的三维定量构效关系(3D-QSAR)。训练集中18个化合物用于建立预测模型,测试集8个化合物(含17号模板分子和新设计4个分子)作为模型验证。所建CoMFA模型的交叉验证系数(R_(cv)^(2))、非交叉验证系数(R^(2))分别为0.525、0.974,说明所建模型具有较强的稳定性和良好的预测能力。该模型中立体场、静电场贡献率依次为69.9%、30.1%,表明影响抗乳腺癌活性(S_(M))的主要因素是取代基的疏水性和空间位阻,其次是取代基的库仑力、氢键及配位作用。基于此研究结果,设计了4个具有较高抗乳腺癌活性的新化合物。

幼儿家长亲子体育行为发生的影响因素分析——基于清晰集定性比较方法(csQCA)

该研究运用清晰集定性比较分析方法(csQCA),从多因素协同影响的角度,研究幼儿家长亲子体育锻炼行为发生的多因素组合路径。结果表明:(1)当家长有闲暇时间,又有免费运动场所的时候,虽然无人组织指导,只要锻炼项目不难完成,亲子锻炼行为依然会进行;(2)虽然家庭月收入不高,但如果有人组织指导,并且有免费的运动场所,再加上家长有运动兴趣,亲子锻炼行为也会发生。而在所有的影响因素中,有免费的运动场所是影响亲子锻炼行为的核心因素。

Development of a quantitative RT-PCR system for the specific detection of human heat shock mRNA expression

Heat shock proteins (HSPs), as molecular chaperones, play an important role under physiological condition and in the course of many diseases. It would therefore be valuable to determine the expression of cellular hsp gene quantitatively. Using DNA recombinant technique and in vitro transcription system, a complex internal control RNA has been prepared. After opti-