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.     

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.     

环介导等温扩增技术对牛奶中沙门氏菌快速检测的研究

环介导等温扩增技术(LAMP)是一种新型的基因扩增方法,它具有高特异性、高灵敏度、便捷、成本低等特点,因而广泛应用于临床检测.本实验以沙门氏菌高度保守基因fimY基因为靶基因设计两对引物(F3&B3、FIP&BIP),建立LAMP反应体系.经试验分析对体系特异性、Mg2+浓度、反应时间、反应温度进行了优化和摸索,对35株临床菌株均检出达100％,其他阴性菌株基因组均无检出,在人工添加样品检测得其在牛奶中灵敏度为33CFU/管.实验结果表明,该方法耗时少、具有高特异性,适合牛奶中沙门氏菌污染的快速检测.     

环介导等温扩增技术(LAMP)快速检测转基因玉米LY038

为了施行对转基因作物的监管,许多国家和机构制定了转基因标识制度的相关条例,因此建立针对转基因产品的检测方法是十分必要的.本研究运用环介导等温扩增技术(LAMP)建立了对转基因玉米(Zea mays L.) LY038外源基因的快速检测技术,该方法依据cordapA基因的序列设计了4条特异性引物,结果表明,引物特异性良好,该检测体系在63℃恒定温度下,反应50 min,可检测到0.01％的样本,是常规PCR方法的5倍.该检测方法具有高度的特异性、灵敏性和稳定性,该方法不需要特殊仪器、快速简便,在基层和现场检测中有很好的应用前景.     

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.     

环介导等温扩增联合横向流动试纸条可视化检测海豚链球菌方法的建立

海豚链球菌(Streptococcus iniae)是一种革兰氏阳性球菌,呈β溶血,可感染多种淡水和海洋鱼类。本研究利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)进行核酸扩增,通过横向流动试纸条方法 (lateral flow dipstick,LFD)实现检测,建立了一种可应用于海豚链球菌快速检测的LAMP-LFD技术。该技术以海豚链球菌促旋酶B亚单位(gyrase subunit B,gyr B)基因为检测靶标,设计3对引物进行由生物素标记的LAMP扩增反应,产物经异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针杂交后,在LFD上完成检测。经优化的核酸扩增最适条件为65℃反应30 min,在此条件下,阳性扩增起始时间与模板浓度之间呈典型的线性相关性。从核酸扩增反应开始到LFD显色,整个检测时程只需40 min左右,比常规PCR技术缩短约2 h。LAMP-LFD能特异性地检出海豚链球菌,针对病原纯培养物的检测灵敏度为8.70×101cfu/m L,是LAMP检测的10倍、常规PCR检测的100倍。以灵敏度浓度(8.70×101cfu/m L)的海豚链球菌基因组DNA为模板进行的LAMP-LFD结果显示该方法具有良好的重复性。针对人工污染花鲈(Lateolabrax japonicus)肝组织的检测灵敏度为4.35×103cfu/m L,同样为常规PCR检测方法的100倍。利用本方法可成功从患病花鲈的组织样品中检测出海豚链球菌,检测结果与常规的细菌分离鉴定方法结果一致。因此,利用LAMP-LFD能特异、准确、高效地检测出海豚链球菌,而且操作简单、费用低、耗时短,有望成为海豚链球菌的常规检测方法。     

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。     

荧光显色在环介导等温扩增（LAMP）检测猪繁殖与呼吸综合征病毒的应用

介绍了一种应用荧光显色的猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)反转录环介导等温扩增(RT-LAMP)的检测方法.该方法使用了针对靶序列M+N基因的一组引物,并且能在63 ℃等温条件下30 min内完成反应.在敏感性检测中RT-LAMP与RT-PCR都能检测103个包含靶基因片段的重组质粒.几种其它病原的核酸也被用来比较这两种方法的特异性,两种方法只能检测PRRSV.在荧光显色剂应用试验中,证实0.05 mmol/L钙黄绿素以及0.6 mmol/L锰离子可以用于LAMP扩增产物的判断,并且其判断结果与浊度判断结果一致.在对103个临床血液样本的检测中,RT-LAMP与RT-PCR检出的阳性样本数量分别是56个和48个.结果说明,应用荧光显色剂的RT-LAMP检测方法可以快速检测PRRSV.     

转基因玉米LY038转化事件的特异性检测

转基因高赖氨酸玉米LY038在畜禽饲料中已得到广泛应用,但目前还没有关于其侧翼序列扩增及转化事件特异性定性PCR检测方法方面的报道.本研究采用修饰接头连接PCR(modified adapter-linked PCR,M-AL-PCR)技术获得了转基因玉米LY038的外源基因与玉米基因组之间的5′端侧翼序列.据此侧翼 序列设计其转化事件特异性引物,建立了转基因玉米LY038转化事件特异性定性检测方法,扩增片段175bp.以转基因玉米(Zea mays L.)LY038、MIR604、Bt176、Bt11、MON810、MON863、GA21、NK603、非转基因玉米、转基因水稻(Oryza sativa L.)Cry1C*、Cry2A*、转基因大豆(Glycine max)Roundup Ready和转基因油菜(Brassica campestris L.)GT73为材料,验证该方法具有高特异性及灵敏度,最低检测限约为0.1%.研究结果提示,该定性检测体系可准确、快速、高效的检测转基因玉米LY038及其产品.     

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.     

模拟降雨条件下污染土壤中重金属元素径流迁移特征

探明污染土壤中重金属元素随地表径流迁移特征,对于防控重金属污染物的迁移扩散具有重要意义。通过室内模拟降雨试验,研究不同降雨强度下(55,100,120mm/h)污染土壤中Cd、Pb和Cr随地表径流迁移的特征。结果表明:径流中3种重金属元素总量在产流的前20min内逐渐降低,随后趋于稳定;产流初期(产流10min内)颗粒态重金属占径流重金属总量的比例均在80%以上;随产流时间延长,颗粒态Cd和Cr对径流重金属的贡献率快速降低,其颗粒态/溶解态的比值范围分别为47.0～0.4和12.9～0.4,而Pb则主要以颗粒态的形式存在,溶解态Pb对其随径流迁移的贡献率可忽略不计;随降雨强度增大,径流中总Cd和Pb含量显著降低,而径流中总Cr含量以及溶解态重金属含量无明显变化规律。     

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%。     

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.     

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.     

NH4+-K+ co-loaded clinoptilolite as a binary fertilizer

ABSTRACT

Understanding the potential of clinoptilolite (CLI) for adsorption of NH₄⁺ and K⁺, providing appropriate fertilizer formula, and evaluation of the produced zeolitic nutrient sources (ZNSs) to meet the plant need are the main objectives of this study. Three ZNSs (NH₄⁺-saturated, K⁺-saturated and dual-purpose NH₄⁺-K⁺ saturated CLI) were produced, assessed, and compared with commercial N and K fertilizers (CFs) on corn growth in a greenhouse. The results indicated that CLI can potentially adsorb both NH₄⁺ and K⁺ to the maximum values of 25.00 mg-NH₄⁺ g^?1 and 47.61 mg-K g^?1, respectively, and chemisorptions mainly followed the process of adsorption. Saturation of zeolite by NH₄⁺ and K⁺ occurred after 10 and 15 d which lead to ZNSs with 2% and 5% of N and K, respectively. NH₄⁺-K⁺ saturated CLI contained 1% N and 1% K. The greenhouse experiment showed no significant difference between ZNSs and CFs on plant growth. However, the application of both N and K in the form of zeolitic sources significantly increased the uptake of N by the plant. The highest uptake of K (2.05 g pot^?1) occurred in plants supplied with both natural zeolite and CFs. The present results may benefit the future utilization of ZNSs in environmental friendly farming practices.     

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。     

氨甲喋呤(MTX)的直接碘标记方法研究

本文报道了~125I(~131I)抗肿瘤药物氨甲喋呤的制备方法,用直接碘化法——NBS、ChT、Iodogen进行标记,并对这几种方法进行了比较。结果表明,NBS法最好,标记率达90—99％。经Sephadex G-75柱凝胶过滤后得到纯产品,放化纯度为95.4％。所制得的标记产品适用于示踪研究。