RNA实时荧光核酸恒温扩增检测技术 检测解脲脲原体的Meta分析

目的评估RNA实时荧光核酸恒温扩增(simultaneous amplification and testing,SAT)检测技术检测泌尿生殖道拭子及尿液样本中解脲脲原体(Ureaplasma urealyticum,UU)阳性率。方法检索中国学术期刊全文数据库(CNKI)、万方数据库、维普数据库、PubMed、ScienceDirect和Cochrane图书馆等,收集有关探讨实时荧光核酸恒温扩增检测UU阳性率的所有文献,检索起止时间为1990年1月至2018年8月。拟定入选标准和排除标准,2名研究者分别独立进行文献资料提取及文献质量评估,采用Stata 13.0软件进行Meta分析。结果共检索出57篇文献,依据入选标准和排除标准,纳入了21篇文献进行分析,包含了33 176例尿液及拭子样本量,所有标本均运用实时荧光核酸恒温扩增对检测样本进行UU检测。Meta分析结果显示,实时荧光核酸恒温扩增检测UU阳性率的汇总检出率(95%CI)为0.44 (0.39-0.49)。结论实时荧光核酸恒温扩增检测UU具有快速、灵敏的特点,很好的判断预后效果,适合临床实验室泌尿生殖道患者UU检测,值得临床进一步推广应用。     

男性患者解脲脲原体三种不同检测方法评价及药敏分析

目的对男性患者解脲脲原体进行三种检测方法的对比研究及药敏分析。方法选取桂林医学院附属医院自2015年1月至2015年12月收治的406例男性解脲脲原体感染患者作为临床研究对象,分别同时用培养鉴定法、DNA实时荧光定量法(RT-PCR)及RNA实时荧光恒温扩增检测(SAT)法三种方法对标本进行检测。以培养鉴定法作为对照组,对比RT-PCR及SAT方法对解脲脲原体的阳性检出率。结果培养鉴定法、RT-PCR、SAT法阳性检出率分别为41.7%、35.5%、43.6%,培养鉴定法与SAT法阳性检出率明显高于RT-PCR,具有显著差异(χ~2=4.35,P=0.0362);培养鉴定法与SAT法阳性率无明显差异(χ2=2.89,P=0.0982),有较好的一致性(K=0.890,P0.05);药敏结果显示,解脲脲原体对强力霉素、美满霉素敏感性较高,敏感率为97.6%。结论对男性疑似解脲脲原体感染患者建议选择不同检测方法诊断以保证阳性检出率,可以更好指导临床抗生素合理使用。     

酶联免疫吸附试验夹心法检测解脲脲原体方法的建立

目的为了提高解脲脲原体(Ureaplasma urealyticum,UU)检测的快速性。方法用酶联免疫吸附试验(ELISA)夹心法检测UU抗原并与传统的培养法相比较。结果ELISA夹心法敏感度为92.6%,特异度为97.4%,最低能够检测出蛋白含量为5～10ng/ml的UU抗原。结论ELISA夹心法是一种敏感、方便、快捷、适合大规模标本检测解脲脲原体的方法。     

解脲脲原体药物敏感性变迁研究

目的:探讨上海地区2008与2012年泌尿生殖道解脲脲原体(Ureaplasma urealyticum,UU)耐药性变化,为临床合理用药提供参考。方法:采用自制UU药物敏感检测试剂对2008年450例和2012年459例患者的标本进行检测,观察UU阳性情况及UU对交沙霉素、氧氟沙星、阿奇霉素和强力霉素的药物敏感性。结果:2008年分离到150例UU阳性标本和2012年分离到134例UU阳性标本分别对交沙霉素、强力霉素、氧氟沙星和阿奇霉素等4种抗菌素的敏感率有显著性差异;2008年46例和2012年38例男性患者阳性标本中分离的UU分别对交沙霉素和阿奇霉素敏感率有显著性差异;对强力霉素和氧氟沙星敏感率无显著性差异。2008年104例和2012年96例女性患者阳性标本中分离的UU分别对交沙霉素、强力霉素、氧氟沙星和阿奇霉素等4中抗菌素的敏感率都有显著性差异。结论:5年间UU药物敏感性发生了变迁;临床医师应该关注本地区药物敏感性变迁,合理选用抗生素。     

慢性阴道炎患者生殖道解脲脲原体和沙眼衣原体感染分析

目的通过荧光定量聚合酶链反应(PCR)技术检测慢性阴道炎患者阴道内解脲脲原体(Uu)和沙眼衣原体(Ct)感染状况,用以指导临床正确治疗。方法应用荧光定量聚合酶链反应对380例慢性阴道炎患者的阴道分泌物进行解脲脲原体和沙眼衣原体PCR检测。结果解脲脲原体阳性率为42.9%,其阳性标本的平均拷贝数为3.4×105copies/ml,沙眼衣原体阳性率为16.6%,其阳性标本的平均拷贝数为5.8×104copies/ml,解脲脲原体和沙眼衣原体合并感染的阳性率为12.6%。结论PCR技术具有简便、快捷、准确的优点,是目前快速诊断慢性阴道炎患者Uu、Ct感染状况的可靠诊断方法之一。     

男性不育患者解脲支原体感染状况及其对精液参数的影响

目的:观察男性不育患者解脲支原体UU感染状况及其对精液参数的影响,分析治疗前后精液参数的变化.方法:用培养法检测4300例男性不育患者精液UU感染情况并行精液常规检测,与正常对照组进行比较.对UU感染患者根据药敏结果进行治疗,并观察治疗前后精液参数的变化.结果:男性不育患者UU的感染率为39.3％,对照组为12.8％,差异有统计学意义(P＜0.05).男性不育UU阳性患者精子浓度、前向运动PR、正常形态率与同组阴性者比较差异无统计学意义(P＞0.05),与对照组阴性者比较,差异有统计学意义(P＜0.01),与对照组阳性者比较,差异有统计学意义(P＜0.05).经治疗后患者精子浓度、前向运动、正常形态率有明显改善.结论:解脲支原体感染是引起男性不育的主要原因,对精子浓度、前向运动、正常形态率有一定的影响,经过合理治疗后精液质量可以改善.     

乌鲁木齐女性泌尿生殖道支原体感染状况及其 耐药性探讨

目的探讨新疆乌鲁木齐市女性泌尿生殖道感染支原体的状况及其耐药情况。方法采用支原体分离培养药敏试剂盒检测2013年7月至2017年8月间7 899例在本院首次就诊的女性患者的泌尿生殖道分泌物样本,分析支原体培养及药敏结果。结果 7 899例女性泌尿生殖道样本中,支原体阳性1 913例,总阳性率为24.22%,其中单纯解脲脲原体(MH-UU+)1 772例,占支原体感染阳性构成比92.63%。民族中,汉族、维吾尔族、回族、哈萨克族、蒙古族中支原体感染率分别为24.18%、22.85%、24.34%、27.94%、24.21%,差异无统计学意义(P0.05)。单纯人型支原体(MH+UU-)感染对左氧氟沙星、氧氟沙星的耐药率均高于MH-UU+与解脲脲原体和人型支原体混合感染(MH+UU+)(P0.05)。MH+UU+感染对司巴沙星的耐药率高于MH-UU+和MH+UU-感染(P0.05)。结论本地区女性泌尿生殖道支原体以MH-UU+感染为主,首选抗生素为交沙霉素,目前在这5个民族感染的支原体体外抗生素耐药性差异不明显。     

环介导等温扩增技术对解脲脲原体的临床检测

目的建立并优化环介导等温扩增(LAMP)技术对解脲脲原体(U.urealyticum)的检测,并应用于临床样本分析。方法针对U.urealyticum的urease基因设计LAMP引物;研究LAMP的最适温度、最佳检测时间及灵敏度和特异度;与传统PCR检测进行方法学比对。结果 LAMP技术检测U.urealyticum的最适温度和最佳时间分别是61℃和60 min,并且具有良好灵敏度和特异度,较普通PCR检测的灵敏度高出1 000倍。临床样本检测中,PCR和LAMP技术达到的灵敏度分别为25.00%和87.50%。两种方法的特异度均为100.00%。结论 LAMP与PCR相比在基层检测和大规模筛查方面有显著的优势和巨大的利用价值。     

运用PCR 扩增技术检测解脲脲支原体生物群的临床应用

目的:探讨运用酶链聚合反应(PCR)技术检测泌尿生殖道解脲脲原体(Uu)的生物群,分析Uu生物群与临床症状的相关性。方法:以支原体16S rRNA保守区域基因为扩增靶序列设计引物,采用PCR方法扩增Uu 16S rRNA基因检测125例临床标本,并将检测结果与临床症状进行相关性分析。结果:PCR法检出Uu阳性率44.8%,其中35例Uu生物1群阳性,其中有16例有症状;27例Uu生物2群阳性,其中有18例有症状。Uu生物1群感染与症状的相关性无统计学差异(P>0.05),而Uu生物2群感染与症状相关性有统计学差异(P<0.05)。结论:PCR检测Uu 16S rRNA基因可用于Uu生物群的检测,Uu生物2群可能是非淋菌性尿道炎(NGU)的一个致病菌,而Uu生物1群与NGU无明显相关性。     

反相斑点杂交法对解脲脲原体分型的研究

目的研究以聚合酶链反应为基础的快速检测与鉴定解脲脲原体基因型的方法。方法选择2003年11月至2005年11月在中山大学附属第二医院门诊就诊的有外阴阴道炎症状和体征的患者601例,设为病例组,同期无自觉症状的正常体检人群306例,设为对照组,分别取宫颈分泌物待检测。将解脲脲原体不同基因型的特异探针固定在硝酸纤维素膜上,临床标本按常规方法提取解脲脲原体DNA,采用生物素标记的解脲脲原体特异通用引物PCR扩增DNA,然后分别与解脲脲原体不同基因型特异探针杂交、显色。结果病例组解脲脲原体阳性421例占70.0%,对照组解脲脲原体阳性126例占41.2%。病例组中单型别感染的U.parvum占65.4%,其中1型、3型、6型和14型分别占28.8%、43.3%、26.0%和1.9%,U.urealyticum占18.4%;对照组中单型别感染的U.parvum占79.3%,其中1型、3型、6型和14型分别占63.2%、21.1%、15.7%和0.0%,U.urealyticum占13.8%。18例阳性标本随机DNA测序鉴定,均为相应的解脲脲原体基因型。结论U.parvum群,尤其是其中的1、3、6型别是正常人群携带的可能性较大,U.urealyticum则有可能和1型起协同作用或独自导致疾病。用反相斑点杂交进行解脲脲原体基因分型,方法简单、实用,适用于临床。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Effect of soil salinity and water stresses on growth and content of nitrogen,chloride and phosphate of wheat and triticale

Summary Three wheats and one triticale were grown, up to flowering stage, in pots on calcareous soil adjusted to a range of salinities (S₁=3.5, S₂=6, S₃=8.5, and S₄=11 mmhos/cm, 20°C, soilpaste extract) by adding solution consisting of 3∶2∶1 of Na-, Ca- and Mg chlorides in chemical equivalent amounts. Moisture in the pots was kept at 100% (W₁), 40% (W₂) and 20% (W₃) of the available water. The vegetative growth, nitrogen and phosphate were affected by S and W treatments, chloride was affected only by S. The interaction S×W affected only dry weight. Varietal effect was observed between wheat as a group and triticale. Multiple quadratic regression equations of these properties on salinity and water revealed that the higher the available water the wider the range of tolerable salinity. Triticale was relatively more tolerant to water stress. Salinity increases Cl and decreases N, whereas water stress enhances N accumulation to a certain extent. However, in triticale at S₃ and S₄ the effect of water stress on N was overshadowed by the excessive salinity. This did not occur for the wheat (Florence). P trends were described. R² for P was low (0.7435–0.3603) which made interpretations rather difficult.     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭示草地放牧演替的生理机制有重要意义.大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段.放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势.牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关.放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关.