大鲵致病性嗜水气单胞菌SYBR GreenⅠ荧光定量PCR诊断试剂盒的研制

根据GenBank中致病性嗜水气单胞菌特异性的气溶素基因序列,设计1对引物,利用普通PCR技术扩增获得hlyA基因片段,并克隆到pMD-18T载体上作为阳性标准品。通过对SYBR GreenⅠ荧光定量PCR反应条件的优化,建立了快速检测致病性嗜水气单胞菌的SYBR GreenⅠ荧光定量诊断方法,以此为基础研制出试剂盒。试剂盒扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,对非致病性嗜水气单胞菌、弗氏柠檬酸杆菌、迟缓爱德华菌、柱状黄杆菌均无阳性信号扩增,重复性好,灵敏度可达1.0x101拷贝/uL。结果表明研制的致病性嗜水气单胞菌SYBR GreenⅠ实时荧光定量PCR试剂盒具有特异、灵敏、快速、重复性好等特点,适合于大鲵临床样品的检测。     

山茶根结线虫的LAMP-LFD快速检测方法

为了提高检测效率,本研究利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)扩增核酸,用横向流动试纸条(lateral flow dipstick,LFD)检测产物,建立了一种山茶根结线虫(Meloidogyne camelliae)的LAMP-LFD快速检测新技术.以山茶根结线虫核糖体DNA内转录间隔区(ribosomal DNA-intemal transcribed spacer,rDNA-ITS)为检测靶标,设计3对特异性引物进行生物素标记的实时荧光LAMP反应,优化后的LAMP扩增条件为63℃反应15 min.比较LFD、实时荧光曲线以及琼脂糖凝胶电泳等3种产物检测方法,结果表明,LAMP产物与异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针ITS-HP杂交5 min后即可在LFD上显色,从LAMP反应开始到LFD结果判断仅需25min,比常规PCR技术缩短约2h.LAMP-LFD技术能特异性地检测山茶根结线虫,对其基因组DNA的检测灵敏度为4 pg/μL,低于常规PCR方法100倍;对其单条2龄幼虫(J2)检测灵敏度为1/1 000条线虫.本研究建立的快速、灵敏、特异性LAMP-LFD检测技术可应用于山茶根结线虫的口岸检验.     

荔枝霜疫霉巢式PCR和LAMP检测方法的建立

由荔枝霜疫霉(Peronophythora litchii)引起的荔枝霜疫霉病是荔枝(Litchi chinensis)生产上的重要病害,建立霜疫霉快速准确检测方法,对于该病的早期诊断和及时防控至关重要.本研究以荔枝霜疫霉三磷酸鸟苷(GTP)结合蛋白基因(GTP-binding protein gene,Ypt1)为靶序列,设计特异性引物,建立了巢式PCR和环介导等温扩增(loop-mediated isothermal amplification,LAMP)两种检测方法,并进行特异性和灵敏度验证.特异性检测表明,只有不同来源的21个荔枝霜疫霉菌株经PCR扩增获得249 bp的特异性条带,同时,在钙黄绿素指示剂的作用下,LAMP检测显示绿色,且扩增产物用2.0％琼脂糖凝胶电泳出现特有的梯形条带,而其他13种不同卵菌近缘种及8种常见病原真菌42个菌株均未观察到这些现象.灵敏度检测显示,将特异引物PvYF1/PvYR1与疫霉属Yptl通用引物Yph1F/Yph2R进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达100fg/25 μL,较常规PCR提高1 000倍,而LAMP方法检测灵敏度是巢式PCR的10倍.本研究分别采用常规PCR、巢式PCR和LAMP方法对采集自福建省荔枝霜疫霉病典型症状及可疑症状的荔枝叶片或果实进行检测,并用传统分离方法进行验证.结果表明,在漳州采集的30份样品中,常规PCR、巢式PCR和LAMP方法的阳性检出率分别为17/22(77.3％)、20/22(90.9％)和21/22(95.5％);在莆田采集的25份样品中,常规PCR、巢式PCR和LAMP方法的阳性检出率分别为15/17(88.2％)、16/17(94.1％)、17/17(100％).可见LAMP方法明显提高了检测效率,并且具有检测程序便捷,所需设备简单和肉眼能判断结果的优势,适合基层部门及田间荔枝霜疫霉快速检测.     

马铃薯卷叶病毒RT-LAMP检测方法的建立

为建立一种快速准确检测马铃薯卷叶病毒(potato leafroll virus,PLRV)的方法,本研究以PLRV CP(coat protein,CP)基因ORF3保守序列为靶标,建立了PLRV反转录环介导等温扩增(RT-LAMP)检测体系,并对建立的RT-LAMP检测体系的特异性、灵敏度、实际应用效果进行评估。结果表明,建立的PLRV RT-LAMP检测方法,在62℃恒温下扩增50 min,扩增产物中加入双链嵌合荧光染色(SYBR Green Ⅰ),阳性样品颜色变为绿色,阴性样品颜色仍为褐色,可直接目测判断待检测样品是否感染PLRV。该方法只特异性检测PLRV,不与马铃薯X病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯S病毒(PVS)、马铃薯A病毒(PVA)和马铃薯M病毒(PVM)等其他5种马铃薯主要病毒发生交叉反应。此外,建立的PLRV RT-LAMP检测方法的灵敏度较反转录PCR(RT-PCR)方法高100倍。田间样品检测验证,该方法与标准RT-PCR方法符合率达100%。本研究建立的以SYBR Green Ι为颜色指示的PLRV可视化检测RT-LAMP方法,特异、灵敏、便捷,成本低,可满足科研、基层单位对该病毒快速诊断和检测的需要。     

西尼罗病毒荧光定量PCR检测体系的建立及评价

本研究建立了一种实时荧光定量PCR快速核酸检测西尼罗病毒的方法。通过序列比对和blast分析，确定西尼罗病毒Caspid蛋白保守区基因为检测的目的基因，引物采用Primer Premier5.0软件进行设计。本研究建立的检测方法利用SYBR Green染料，相比探针成本较低。通过溶解曲线分析表明，建立的检测方法在扩增过程中没有发现有二聚体的产生。本检测体系在用空白对照及类似的乙脑病毒作为扩增对照时，没有发现非特异性产物的生成，表明该体系对于西尼罗病毒的检测是特异的。将阳性对照标准品进行10倍梯度稀释后可检测到102copies/μL样品，表明该检测体系具有较高的检测灵敏度。     

对虾白斑综合征病毒可视化环介导等温扩增(LAMP)检测技术的建立与应用

针对当前对虾(Penacus orientalis)养殖业亟需研究开发一种简便快速、敏感特异的检测技术来实现对对虾白斑综合征病毒(White spot syndrome virus,WSSV)进行检测监控的现状,本研究根据WSSV基因组ORF120保守区序列,设计合成6条环介导等温扩增(loop mediated isothermal amplification,LAMP)特异性引物,分别为外引物F3/B3、内引物FIP/BIP和环引物LF/LB。利用钙黄绿素/氯化锰作为检测指示剂,通过对反应条件优化,建立了可视化WSSV-LAMP检测方法。在检测中未发生扩增反应时,钙黄绿素被氯化锰螯合,呈淬灭状态。当样品中有WSSV靶基因存在时,大量DNA被合成,并产生同样大量的焦磷酸根离子,此时焦磷酸根离子竞争性与Mn2+结合,形成稳定的不溶性焦磷酸盐沉淀,钙黄绿素被释放而发出肉眼可见的黄绿色荧光。该扩增一次性反应约60min即可完成对待检样品的检测。经测定,本方法能特异性地检测出阳性样品中的WSSV目的基因,并能通过扩增产物所产生的黄绿色变化与阴性样品区别;本技术对目的基因的最低检测量为1fg。在临床检测试验中,WSSV-LAMP与世界动物卫生组织(OIE)推荐的WSSV-PCR检测方法均能从250份对虾样品中,同时检测到15份编号相同的阳性样品,符合率达100%。除此之外,WSSV-LAMP还能从另外3份PCR检测为阴性的样品中检测到目的基因。比较这两种方法的阳性检出率,WSSV-LAMP为7.2%(18/250),WSSV-PCR为6.0%(15/250),WSSV-LAMP对自然感染的临床样品阳性检出率要比WSSV-PCR高。说明WSSV-LAMP在病毒含量较低的临床样品检测中比OIE推荐的PCR方法具有更高的技术优势,能在对虾养殖生产上的临床诊断、育种及口岸检疫,甚至海洋生态监测中对WSSV进行现场检疫。     

牛支原体的环介导等温扩增快速检测技术研究

牛支原体(Mycoplasma bovis是感染牛的一种重要的致病性病原体.本研究利用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术建立了牛支原体的快速检测方法.该方法以牛支原体保守性基因uvrC为靶序列设计了5条特异引物,在58℃等温条件下,60 min即可完成反应.LAMP方法能检测出20 pg牛支原体DNA,较PCR方法高100倍,用牛鼻支原体(M.bovirhinis)、无乳支原体(M.agalactiae)、精氨酸支原体(M.ariginini)、牛副流感病毒(BPIV)、牛腺病毒(BADV)、牛传染性鼻气管炎病毒(IBRV)作特异性检测,两种方法均有很高的特异性.本研究还将荧光显色剂钙黄绿素(calcein)和Mn2+用于LAMP反应结果的可视化检测.临床鼻拭子样品煮沸后,可直接进行等温扩增,省去了DNA提取步骤.在对167个临床鼻拭子样本的检测中,LAMP方法检测结果阳性率(26.95％)高于PCR方法检测出阳性率(19.16％),这证明本研究所建立的方法,与PCR法比较更适于临床样品的检测.研究结果表明,LAMP方法具有操作简便、快速、高效、敏感、特异、经济等特点,适于基层实验室监测的广泛使用.     

利用环介导等温扩增技术快速检测水产品中的副溶血弧菌

副溶血弧菌(Vibrio parahaemolyticus)是一种重要的食源性致病菌。首次将环介导等温扩增技术（Loop-Mediated Isothermal Amplification, LAMP）应用于副溶血弧菌的快速检测。针对副溶血弧菌不耐热溶血毒素基因（tlh）设计四条特异性引物,建立了副溶血弧菌LAMP检测方法,1 h即可完成。对12种细菌共28株菌进行LAMP扩增,仅副溶血弧菌得到阳性结果,证明引物具有很高的特异性;副溶血弧菌基因组DNA和纯培养物的检测灵敏度分别为90 fg/LAMP反应体系和24 cfu/mL,对模拟食品样品进行直接检测,检测限为89 cfu/g;对40份水产样品进行检测,8份副溶血弧菌LAMP阳性,其中6份传统培养阳性。本研究表明,该方法检测副溶血弧菌特异性强、灵敏度高,并且操作简便、快捷、检测成本低,有望发展成为快速检测副溶血弧菌的有效手段。     

二温式多重PCR检测对虾的白斑综合征病毒和桃拉综合征病毒

根据多重PCR的技术原理,利用对虾(Penaeus vannamei)白斑综合征病毒和桃拉综合征病毒的基因序列分别设计了两对特异引物,并将常规三温式PCR扩增程序简化为2个温度梯度,建立二温式多重PCR技术用于对虾白斑综合征病毒(White spot syndrome virus,WSSV)和桃拉综合征病毒(Taura syndrome virus,TSV)的复合快速检测。利用二温式PCR能特异地扩增出WSSV和TSV的基因片段。结果表明,二温式多重PCR技术具有较高的特异性和敏感性,最低能检测到WSSV核酸模板10pg,TSV核酸模板100pg,且对其它一些对虾病原呈阴性。     

Silicon Effect on Nutrient Acquisition of Peanut (Arachis hypogaea L.) Under Aluminum Stress

Aluminum (Al) toxicity represents one of the main yield-limiting factors for crops in acid soils. Silicon (Si) is known to increase tolerance in higher plants. This study was conducted to determine whether treatment with Si could improve nutrient uptake by peanut under Al stress. Peanut (Arachis hypogaea L. cv Zhonghua 4) was raised with or without Si (1.5 mM) in the growth chamber under 0 and toxic Al (0.3 mM) levels. Aluminum stress significantly decreased the root- and total-dry weight by 52.4% and 32.0%, respectively. The content of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) was significantly decreased, but that of Al increased markedly in shoots and roots after Al exposure at seedling, flower-needle, and pod-setting stage. Silicon alleviates Al toxicity in peanut plants in relation to Al distribution and allocation of tissue P, K, Ca, and Mg by favoring the partitioning of dry mass to roots.     

Trace Metal Contamination During Grinding of Plant Samples

Inductively coupled plasma-mass spectroscopic (ICP-MS) analysis of leaves from 22 cabbage crops in the Sa P? and B?c Hà districts of Láo Cai Province, North-Western Vi?t Nam, revealed unexpectedly high concentrations of chromium (Cr) and nickel (Ni). The concentrations were strongly linearly related (r² = 0.94), indicating sample contamination during grinding through a stainless-steel hammer mill. We tested this hypothesis in two ways. First, brown rice ground through the same mill was contaminated not only by Cr and Ni, but also cobalt (Co), iron (Fe) and molybdenum (Mo). Second, scanning electron microscopy/energy dispersive analysis of x-rays (SEM/EDS) of the ground samples revealed small fragments with co-located Fe, Cr and Ni, consistent with stainless steel wear fragments. Other grinders may perform differently and we suggest that quality assurance protocols for trace metal analysis of plants should include testing for grinder wear metals. Lastly, brown rice appears to be convenient for investigating contamination of plant tissues during grinding.     

Effects of humic acid on remediation of the nutritional deficiency of gerbera in hydroponic culture

Information is scant on the effect of humic acid (HA) on physiological, antioxidant and photosynthesis attributes of gerbera plants undergoing nutrient deficiency in culture solution. Gerbera plants cv. Malibu were grown in a factorial experiment based on a completely randomized design with 3 replications, using 3 different nutrient solutions [complete nutrient solution (NSc), 25% NSc (NS1), and 50% NSc (NS2)] treated with 2 levels of humic acid [0 (HA0) and 500 mg/l (HA1)].The interaction effect of HA and NS showed that HA improved the flower number in NSc, the transpiration in NS1+HA1, photosynthesis rate in NSc+HA1, stomatal conductance (gs) in NS2, mesophyll conductance of leaves in all NS levels and photosynthetic water use efficiency in NSc+HA1. The interaction effect of nutrient solution and HA on antioxidant activity was inconclusive, malondialdehyde content was the highest in NS2 and the lowest in NS1+HA1. The peroxidase activity increased in complete nutrient solution with and without HA and there were no differences among other treatments. Superoxide dismutase activity increased in NS1 and complete nutrient solution with HA and reached the highest in NSc. Humic acid was more effective in nutrient uptake, i.e., nitrogen, phosphorus, potassium, calcium, zinc, and iron (N, P, K, Ca, Zn, and Fe) in complete nutrient solution compared to NS1 and NS2. Conclusively, humic acid can compensate the nutrient deficiency stress of the culture solution in regards to protein synthesis, photosynthesis attributes regardless of the nutrient uptake of gerbera.     

氮磷钾不同配比对饲用稻威优198产量及糙米蛋白含量的影响

采用田间小区试验研究“推荐配比”(N:P2O5:K2O比率为190:90:100 kg hm-2)、“高氮量配比”(N:P2O5:K2O比率为210:90:100 kg hm-2)、“低氮量配比”(N:P2O5:K2O比率为170:90:100 kg hm-2)以及“常规配比”(N:P2O5:K2O比率为216:112.5:202.5 kg hm-2)4种氮、磷、钾配比施肥对饲用稻威优198蔗糖合成酶(SUS)、腺苷二磷酸焦磷酸化酶(AGPase)、硝酸还原酶(NR)、谷氨酰胺合成酶(GS)以及产量和糙米蛋白质的影响。结果表明:“推荐配比”能提高不同生育时期水稻功能叶(旗叶)和粒籽中碳、氮代谢关键酶的活性,这些关键酶活性的变化显著影响水稻产量和糙米全氮以及蛋白氮的含量。统计(P0.05)结果证实“推荐配比”能提高水稻产量达到8200 kg hm-2,与“常规配比”相比产量提高了24.81%;“推荐配比”糙米全氮和蛋白氮含量分别达到22.70 g kg-1和21.98 g kg-1,与“常规配比”相比差异显著,并且其全氮和蛋白氮含量分别提高17.01%,18.38%。     

小反刍兽疫N蛋白单克隆抗体的制备及竞争ELISA方法的建立

小反刍兽疫(PPR)是由小反刍兽疫病毒(PPRV)引起的一种危害严重的急性接触性传染病。为了检测血清中存在的抗PPRV的抗体,本研究利用纯化后的真核表达的PPRVN蛋白重组蛋白(rBacmid-PPRN)作为免疫原免疫BALB/c小鼠(Mus musculus),并使用原核表达的PPRVN蛋白重组蛋白(rpET-PPRN)作为间接ELISA检测抗原,共筛选得到8株抗PPRVN蛋白的单克隆抗体(mAb)。其中1D5单抗腹水效价达1∶106,分泌单抗亚类为IgG2b。以1D5作为竞争抗体、纯化后的rpET-PPRN蛋白作为包被抗原,建立了检测小反刍兽疫血清抗体的单抗竞争性ELISA(c-ELISA)方法。所建立的c-ELISA方法能够特异性的区分牛瘟阳性血清和小反刍兽疫阳性血清,并具有较高的灵敏度和特异性。应用建立c-ELISA检测方法检测共129份山羊(Capra hircus)血清样品,与标准c-ELISA试剂盒的符合率为96.9%。本研究成功地建立了可特异性检测动物血清中抗PPRV抗体的竞争性ELISA方法,对小反刍兽疫的诊断、疫苗免疫水平的监控及控制其流行具有重要意义。     

四种常规方法提取伊利石有效钾的机制比较

采用化学分析、X射线衍射、中红外光声光谱以及原子力显微镜的方法,比较了0.2 mol L~(-1)四苯硼钠法、1 mol L~(-1)沸硝酸法、2 mol L~(-1)冷硝酸法和2 mol L~(-1)热盐酸法浸提伊利石中有效钾的机制。结果表明,四苯硼钠法浸提时,伊利石中钾素释放量达到全钾量的59.5%,且基本均通过层间交换反应予以释放,结构离子铁、铝和硅释放量极低;采用三种酸溶液浸提时,其钾素释放量仅占全钾量的1.53%~2.46%,通过层间交换反应释放的钾量占释放量的比例为88.4%~94.0%。四苯硼钠浸提时伊利石层间距扩大,产生次生过渡矿物,并形成富硅表层,但在伊利石表面无溶蚀特征;三种酸溶液浸提时伊利石结构无改变,但其结晶度降低,且表面有明显的溶蚀特征。因此,土壤矿物层间钾是作物可利用有效钾的主要来源,三种酸溶液浸提方法一方面低估了有效钾容量,另一方面提取了一部分不能为植物所利用的结构态钾,不适宜于用来评价伊利石及土壤有效钾库容量。     

羟基萘酚蓝在伪狂犬病病毒环介导等温扩增检测方法中的应用

伪狂犬病毒(Pseudorabies virus,PRV)传统诊断方法具有程序繁琐、周期长、成本高、特异性差等缺点,本实验建立了敏感、特异的PRV的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法,并在此基础上应用金属离子指示剂羟基萘酚蓝(hydroxy naphthol blue,HNB)进行反应结果判定.实验选取保守性较高的gB基因(glycoprotein B,糖蛋白gB)筛选引物,确定反应体系后,可在45 min内完成检测.LAMP方法特异性与PCR方法一致,敏感性是PCR方法的100倍,最低能够检测10个拷贝的目的基因.在HNB的应用试验中,发现LAMP反应体系中,HNB浓度为150 μmol/L时,阴阳性的颜色对比明显且反应的敏感性最高,可用于LAMP扩增产物的判断.临床样本的检测结果表明,PCR方法和LAMP方法的检出率一致.因此,本研究中所建立的LAMP方法是一种高度特异敏感的,可替代PCR方法的检测技术.     

Arbuscular Mycorrhizal Fungi Enhance Tolerance of Rosa multiflora cv. Burr to Bicarbonate in Irrigation Water

ABSTRACT

High bicarbonate (HCO₃ ^?) of irrigation water can be detrimental to plant growth in sustainable horticultural production systems. The ability of arbuscular mycorrhizal fungi (AMF), ZAC-19, (composed of Glomus albidum, Glomus claroideum, and Glomus diaphanum) to enhance tolerance to HCO₃ ^? was tested on Rosa multiflora cv. Burr. Arbuscular mycorrhizal colonized and non-inoculated (non-AMF) plants were treated with 0, 2.5, 5, and 10 mM HCO₃ ^?. Increasing HCO₃ ^? concentration and associated high pH and electrical conductivity (EC)—reduced plant growth, nutrient uptake, and acid phosphatase activity, while increasing alkaline phosphatase activity (ALP). Inoculation with AMF enhanced plant tolerance to HCO₃ ^?, as indicated by greater growth (leaf, stem, and total plant dry weight, leaf area and leaf area ratio), leaf elemental concentration [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), iron (Fe), zinc (Zn), aluminum (Al), boron (B)], leaf chlorophyll concentration, higher mycorrhizal inoculation effect, lower root Fe reductase activity, and generally lower soluble ALP activity. While AMF colonization was reduced by increasing HCO₃ ^? concentration, colonization still occurred at high HCO₃ ^? concentration. At 2.5 mM HCO₃ ^?, AMF plant growth was comparable to plants at 0 mM HCO₃ ^?, further indicating the beneficial effect of AMF for alleviation of HCO₃ ^? plant stress.     

产L-苯丙氨酸解氨酶菌种筛选

从土壤中筛选出1株具有L-苯丙氨酸解氨酶(L-Pheny la lan ine amm on la-lyase,PAL)较高酶活力的产生菌株——酵母菌SA 1。L-酪氨酸(L-T yr)为该菌株产PAL的最佳诱导物;利用反式肉桂酸转化L-苯丙氨酸(L-Phe)的最佳酶促反应条件为:2.0%反式肉桂酸,碳酸氢铵∶氨水(W∶V)=4∶4,pH 10.6,34°C,反应8 h,在转化体系中添加甘油对酶活力有较好的保护作用。     

Irrigation Module and Sowing Date Affect Seed Cotton Yield,Quality, Productivity Indices,and Economics of Cotton in North-western India

ABSTRACT

Delayed sowing and imprecise application of irrigation water to cotton has been the major hurdle in sustaining cotton yield in north-western India. Therefore, studies were initiated to ascertain the impact of heavy or normal level of presowing irrigation (PSI), scheduling time of first postsowing irrigation (POSI) under two sowing dates (SDs) on cotton arranged in a split block design replicated thrice. PSI_h (100 mm) recorded 23.2% higher seed cotton yield over PSI_n (70 mm) owing to better yield attributes and higher stand. First POSI at 4 weeks after sowing (WAS) resulted the highest yield (3072 kg ha^?1), while one at 6 WAS (POSI₆) recorded least due to poor population and reduced yield attributes. Water productivity under POSI₆ was lesser by 29.1%, 25.2%, and 16.2% as compared to POSI₃, POSI₄, and POSI₅, respectively. April sown crop out yielded the May sown cotton crop by 939 kg ha^?1 due to better yield attributes. Nitrogen factor productivity (NFP) among SD remained higher by 23.3% for April as compared to May sowing. PSI_h exhibited better NFP over PSI_n. POSI₆ recorded least NFP by 24.4%, 28.5%, and 16.3% as compared to POSI₃, POSI₄, and POSI₅, respectively and was indicative of relatively poor utilization of N under delayed schedules. Therefore, planting in April after heavy PSI and scheduling first POSI at 4 WAS is best strategy for sustaining cotton yield and maximizing farmer profitability.