Determination of vc and mating types of Cryphonectria parasitica isolates by multiplex PCR and their genetic diversity in 13 chestnut-growing provinces of Turkey

Vegetative compatibility (vc) and mating types and genetic diversity of Cryphonectria parasitica isolates were determined using 183 isolates obtained from 215 infected chestnut trees growing in 13 provinces of Turkey. Based on the cultural aspects, 143 of these isolates were evaluated as virulent whereas the remaining 40 isolates were hypovirulent. When vc types of 183 isolates were classically differentiated, 135 of them matched to EU-1 (82.3%), 29 of them to EU-12 (17.6%) vc type, whereas 19 of them did not match to the two. When molecular vic markers were used, all the isolates were assigned to two EU vc types; 149 to EU-1 (81.4%) and 34 (18.5%) to EU-12. Of the majority of the isolates, 134 (73.2%) had mating-type MAT-1, while 44 (24%) isolates had MAT-2 and 5 (2.8%) isolates had both mating types. The population analysis based on two DNA marker systems, Inter-Primer Binding Site and Start Codon Targeted Polymorphism, showed no intraspecific genetic variation among the C. parasitica isolates. The prevalence of two dominant vc types revealed by this study shows that biological control with hypovirulent EU-1 and EU-12 isolates will be significant for the country. The results might be helpful to chestnut breeders carrying out resistance breeding studies to manage this disease based on hypovirulence attributed to Cryphonectria hypovirus 1.     

玉米样品中转基因玉米Bt176含量检测

根据其检测特异性,转基因产品检测技术可分为筛选、基因、构建和转化体特异性检测四类.由于转化体特异性检测方法的具有高度特异性,目前,一些发达国家对于转基因产品检测方法正逐步过渡到转化体特异性序列的检测方法.本研究针对转基因玉米Bt176的外源基因3'端与玉米基因组DNA相接区序列设计具有转化体特异性的引物和荧光探针.利用实时荧光定量PCR,以已知Bt176含量样品为标准样品建立内外源基因标准曲线,利用该标准曲线对多个玉米样品Bt176玉米成分含量进行检测,结果显示该方法检测结果准确,检测特异性强,Bt176玉米检测极限浓度达到0.001 ng/μL.同时该方法操作简单,适合转基因样品大批量检测.     

数字PCR技术在动物疫病检测中的应用

为了支持动物疫病流行地区的控制及消除计划,以及在动物疫病非流行地区进行有效筛查,必须使用更加准确灵敏的诊断手段。数字PCR可实现绝对定量及痕量核酸的检测,与传统检测方法相比具有不依赖参考物或标准品、对抑制剂具有较高耐受性、灵敏度和精准度更高的优点。数字PCR作为一种定量分析的核酸检测新方法,在动物疫病检测中得到了应用,围绕数字PCR技术的原理、应用、存在的问题及发展趋势等进行综述及分析,为数字PCR在动物疫病检测中的进一步应用提供参考。     

禽腺病毒血清4型感染鸡组织中NLRP3基因转录水平的实时荧光定量PCR分析

旨在分析禽腺病毒血清4型(FAdV-4)感染鸡组织中NLRP3基因的转录水平,本研究设计鸡NLRP3特异性引物,利用RT-PCR扩增NLRP3基因180 bp片段并克隆至pMD-18T载体,制备重组质粒pMD-18T-NL-RP3.以pMD-18T-NLRP3质粒作为标准品进行荧光定量PCR并建立标准曲线.通过反应条件...     

黄瓜花叶病毒外壳蛋白基因转化辣椒的研究

以辣椒品种'B162'为试材,探讨了通过根癌农杆菌介导法将黄瓜花叶病毒外壳蛋白(CMV-CP)基因转化辣椒的适宜条件.结果表明,将辣椒带柄子叶进行预培养2 d后,用D600 nm为0.3的根癌农杆菌菌液浸泡7 min,再经过共培养2 d后转移到筛选分化培养基中进行培养,有利于获得辣椒抗性不定芽.通过对获得的10株抗性不定芽进行PCR检测,其中有2株扩增出目的条带,初步证明CMV-CP基因已经转入辣椒不定芽中.     

Temporal shifts in diversity and quantity of nirS and nirK in a rice paddy field soil

Denitrification is an important part of the nitrogen cycle in the environment, and diverse bacteria, archaea, and fungi are known to have denitrifying ability. Rice paddy field soils have been known to have strong denitrifying activity, but the microbes responsible for denitrification in rice paddy field soils are not well known. Present study analyzed the diversity and quantity of the nitrite reductase genes (nirS and nirK) in a rice paddy field soil, sampled four times in one rice-growing season. Clone library analyses suggested that the denitrifier community composition varied over sampling time. Although many clones were distantly related to the known NirS or NirK, some clones were related to the NirS from Burkholderiales and Rhodocyclales bacteria, and some were related to the NirK from Rhizobiales bacteria. These denitrifiers may play an important role in denitrification in the rice paddy field soil. The quantitative PCR results showed that nirK was more abundant than nirS in all soil samples, but the nirK/nirS ratio decreased after water logging. These results suggest that both diversity and quantity changed over time in the rice paddy field soil, in response to the soil condition.     

均匀设计优化毛竹EST-SSR PCR反应体系

本研究以毛竹叶片DNA为模板,利用均匀设计U10（54）表,对毛竹EST-SSRPCR反应体系的TaqDNA聚合酶、Mg2＋、dNTPs及引物浓度4因素5个水平进行优化筛选。优化实验结果表明,毛竹EST-SSRPCR的25μL优化反应体系为：10×PCRBuffer（含Mg2＋）2.5μL、TaqDNA聚合酶1.5U,Mg2＋、dNTPs及引物的最适浓度分别是1.0mmol/L、0.1mmol/L、0.48μmol/L;通过梯度PCR试验筛选得到相应引物的最佳退火温度为52.4℃。对优化出的EST-SSRPCR反应体系进行稳定性检测,结果均能获得丰富、稳定、清晰可辨的DNA谱带。该优化体系为利用EST-SSR分子标记对毛竹进行遗传多样性等相关研究工作提供了基础条件。     

狂犬病病毒SYBR-GreenⅠ实时荧光定量PCR检测方法的建立

狂犬病病毒为不分节段单链负股的RNA病毒,属弹状病毒科狂犬病病毒属。世界上几乎所有国家都有狂犬病发生,狂犬病病毒能够使所有温血动物发病致死,死亡率高达100%。本研究根据GenBank中的狂犬病病毒M基因序列,选择保守区域设计引物,通过对SYBR-GreenⅠ实时荧光PCR反应条件进行优化,建立了用于检测狂犬病病毒的SYBR-GreenⅠ实时荧光PCR方法。结果显示,建立的狂犬病病毒实时荧光定量PCR方法,具有特异性强、灵敏度高、重复性好的优点,是开展狂犬病的临床检测的有力工具。     

黑番鸭血管活性肠肽受体-1基因(VIPR-1)的cDNA克隆及其组织表达特性分析

最近的研究表明,血管活性肠肽(VIP)与家禽的就巢习性密切相关。本研究采用反转录PCR方法从黒番鸭(Cairina moschata)母鸭下丘脑组织中克隆了血管活性肠肽受体(VIPR-1)基因的cDNA序列,长度为1125bp,编码355个氨基酸(GenBank登录号:JN625215)。序列比对结果表明,该序列与家禽(鸡、火鸡、鹌鹑)和哺乳动物(人、小鼠、猪、牛)的基因序列分别有93%~94%和69%~71%的同源性,而相应氨基酸序列的同源性分别为96%和70%~72%。荧光定量PCR结果发现,黒番鸭VIPR-1基因的表达量在产蛋期、就巢期和休产期差异显著(P<0.05)或极显著(P<0.01),就巢期表达量最高,休产期次之,产蛋期表达量最低,表明VIPR-1基因与繁殖阶段变化密切相关;对不同组织VIPR-1的表达量分析发现,VIPR-1在垂体、下丘脑和卵巢中均有表达,其中垂体最多,其次是下丘脑,卵巢中的表达量最低,差异极显著(P<0.01)。研究结果提示,VIPR-1基因具有高度保守性,参与下丘脑-垂体-卵巢轴(特别是垂体)对黑番鸭就巢行为的调控。     

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。