甜菜银叶病菌的PCR检测

本研究用16S23S rDNA间的ITS 序列通用引物L1（5′AGTCGTAACAAGGTAGCCGT3′）和L2 （5′ GTGCCAAGGCATCCACC3）扩增甜菜银叶病菌（Curtobacterium flaccumfaciens pv. betae,Cfb）和其它相近细菌的基因组DNA;并对其PCR产物进行回收、克隆和测序,将所获序列和其它已报道的细菌内源转录间隔区(Internally Transcribed Spacer,ITS)序列进行多重比较后设计出Cfb的特异性引物B1（5′GGCCTCGTGTTGTCCCTTATC3′）和B2 （5′GTCACCAATCAACAACCCGAG3′）。此引物可以从Cfb中扩增出387bp 的特异性片段,而其余参试的21个细菌PCR反应结果均为阴性。该方法可以应用于病害防治工作中的Cfb快速、可靠的检测。     

稻曲病菌的Nested PCR检测

为设计稻曲病菌(Ustilaginoidea virens)专化性PCR引物,测定了1991-2001年采集的多个水稻品种、不同水稻产区的菌株的ITS和5.8S rDNA区序列。U. virens的ITS1、ITS2和5.8S rDNA区域的长度为 624-625bp, 序列高度保守。在与麦角菌科其它种比较的基础上,设计了U. virens专化性嵌合引物。采用PCR方法可以灵敏地检测目标真菌,并且与传统的组织观察结果很好地吻合。这一结果为深入研究稻曲病的侵染规律和建立田间早期诊断技术提供了可能。     

应用PCR方法检测油菜菌核病菌对多菌灵的抗药性

通过保守的寡核苷酸引物B1/B3扩增出油菜菌核病菌MBC^HR和MBC^S菌株的部分β-微管蛋白基因,结果发现编码的198位氨基酸由Clu(GAG)突变为Ala(GCG),表现高水平抗药性。根据MBC^HR菌株的突变设计2个快速检测方法：第一种方法是根据MBC^HR菌株197和198位密码子（GACGAG→GACGCG）形成HhaI酶切点（3'CGCG 5'）,将B1/B3的扩增产物874bp片段酶切成193bp和681bp片段,而MBC^S菌株的PCR产物不被酶切;第二种方法用198位突变密码子作为3'末端碱基设计2个等位基因特异性寡核苷酸引物（ASO）用一地“nested”PCR或直接从基因组DNA扩增。通过PCR扩增和ThaI酶切能直接检测油菜菌核病菌的MBC^HR和MBC^S菌株,所得结果与传统菌落直径法相吻合。     

拮抗辣椒疫病菌的红树内生细菌

<正>随着绿色农业的兴起和可持续发展观念的提出,高效、无毒、无害的植物病虫害防治措施越来越受到人们的关注和重视。生物防治因对人畜安全无毒、无污染、无残留,具有不易使病原菌产生耐药性、生产工艺     

苹果黑星病菌SSR反应体系的优化

通过对苹果黑星病菌基因组DNA的SSR反应中一些重要参数进行优化,结果表明,最适反应体系为:2 5μL体系中,10×Buffer Mg Cl2 2 0 mm ol/ L 2 .5μL ,d NTP 10 0μmol·L- 1 、引物0 .5μmol·L- 1 、Taq DNA聚合酶1.5 U ,DNA模板2 m g·L- 1 ,dd H2 O 19.5μL .PCR扩增程序为93℃,2 min,5 7℃,30 s,1个循环;72℃,1m in,93℃,30 s,5 7℃,30 s,4 0个循环;72℃,10 min.     

栗疫病菌的营养体亲和性基因和dsRNAs对病毒传播的影响

研究了栗疫病菌（Ｃｒｐｈｏｎｅｃｔｒｉａｐａｒａｓｉｔｉｃａ（Ｍｕｒｒ）．Ｂａｒｒ）营养体亲和性基因及ｄｓＲＮＡ病毒对菌株间病毒特征与传播影响,试验选用已知４个ＶＣ基因座位的１５个ＶＣ基因型菌株和３种ｄｓＲＮＡ病毒,通过含病毒菌株与野生型菌株的配对培养,将病毒逐个转入不同ＶＣ基因型菌株,将不同ＶＣ基因型的含病毒菌株与具特定ＶＣ基因差异的野生型菌株配对培养,根据培养两周后野生型菌株培养性状的改变与否     

新疆地区小麦白粉病菌寄主范围的研究

用小麦白粉病菌11个生理小种的混合菌种,对新疆地区的小麦近缘植物的7个属22个种的47份材料进行接种,除6份免疫外,其余均接种成功.用其中6个属19个种的29份小麦近缘植物产生的白粉病菌,对小麦回接,参试的29份材料全部回接成功.小麦白粉病菌对小麦近缘植物的寄生像在小麦上一样,有明显的寄生专化性.感病的小麦近缘植物的78.0%对小麦白粉病菌的感病性,随生育期增长而急剧下降.文中并对小麦白粉病中间寄主的作用进行了讨论.     

小麦根腐病菌索氏平脐蠕孢SYBR Green I 实时荧光定量PCR检测技术研究

由索氏平脐蠕孢Bipolaris sorokiniana引起的小麦根腐病,常和其他土传真菌病害混合发生,传统的症状鉴别方法很难区分,导致病害防控难度增加。为建立病菌实时荧光定量检测体系,根据ITS序列设计引物,筛选出1对特异性引物BS‐F/R,扩增片段大小为280bp。以菌丝DNA为标准品构建实时荧光定量标准曲线,并对其灵敏度、特异性、可重复性进行评价。结果表明,建立的实时荧光定量PCR检测方法速度快,灵敏度高,特异性强,重复性好。构建的荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系,溶解曲线的吸收峰单一,扩增效率良好。利用该定量检测体系,可以检测出田间小麦样品中52.8fg/μL的病菌DNA。     

大豆疫霉根腐病菌的rDNA ITS序列分析

采用真菌核糖体基因转录间隔区(ITS)通用引物,PCR扩增了大豆疫霉根腐病菌具有差异的17个菌株的ITSI与ITS2,经过与DL2000的标准分子量DNA进行比较,得到了大约800~1000bp左右的片段,并对PCR产物进行了序列测定。以USA为外类群利用最大简约法构建了大豆疫霉根腐病菌的系统发生树,并分析了菌株之间的遗传进化关系。结果表明:不同菌株ITS1和ITS2在碱基构成上有很大差异,17个菌株大致分为4个谱系中,且来自于同一地区的菌株大都分布在同一谱系中,显示出地理上的差异。     

简并PCR技术在疾病检测中的应用

普通PCR引物对目的基因扩增的单一特异性,限制了其在临床疾病检测中的通用性范围,而多重PCR需要避免若干引物对之间二级结构的干扰,应用同样受到一定的限制。简并PCR技术中简并性引物是基于基因保守区域的分析并结合简并碱基而设计,可以检测出高度相似的靶基因,具有高效性、灵敏性、特异性和低成本的特点,目前已被广泛应用于多个领域。综述了简并PCR技术在临床、食品安全、动植物疫病、寄生虫、水产养殖等领域疾病检测中的应用,并对其优缺点进行了讨论,以期为简并PCR的进一步发展以及在其他领域的应用提供参考和指导。     

PCR hot-start using duplex primers

A new technique of PCR hot-start using duplex primers has been developed which can decrease the undesirable products arising throughout PCR amplification thereby giving better results than a manual hot-start method.     

New genus-specific primers for the PCR identification of novel isolates of the genus Streptomonospora

The halophilic actinomycete genus Streptomonospora, forming a distinct branch in the 16S rRNA gene phylogenetic tree adjacent to the genera Nocardiopsis and Thermobifida, was first proposed by Cui et al. to accommodate the species type Streptomonospora salina. During a biodiversity and taxonomic study on halophilic filamentous actinomycetes from a saline lake in western China, numerous new halophilic actinomycetes strains were isolated. To confirm whether they are members of the genus Streptomonospora, one set of genus-specific oligonucleotides was designed which allows rapid detection of members of the genus Streptomonospora by means of PCR amplification. The genus specificity of these primers was validated with reference strains as well as with wild-type isolates, which exhibited morphological characteristics common to this genus.     

Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence

Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.     

紫云英ISSR引物的筛选及PCR反应体系的优化

以紫云英为研究材料,用哥伦比亚大学(UBC)公布的100条ISSR引物和11种株系紫云英品种的DNA为模板进行PCR扩增,筛选出33条扩增条带较好的ISSR引物,对其中的ISSR引物进行梯度PCR,筛选出最佳的退火温度。再采用正交试验和单因素试验相结合的方法对紫云英ISSR-PCR反应体系的5种因素(模板、Mg2+、TaqDNA聚合酶、dNTP及引物)进行优化浓度。确立了适合紫云英的ISSR分析的反应体系。在25μl反应体系中,其反应浓度为:DNA模版50.00ng,Mg2+2.00mol/L,Taq聚合酶1.0 U,dNTP 0.25mmol/L,引物0.20μmol/L,2.5μl 10×buffer。本试验为以后利用ISSR技术进行紫云英遗传多样性分析和物种保护奠定了技术基础。     

Universal primers for the PCR-mediated amplification of DNA 1: a satellite-like molecule associated with begomovirus-DNA beta complexes

DNA 1 is a single-stranded DNA molecule of approximately 1370 nucleotides. It is associated with monopartite geminiviruses of the genus Begomovirus, which require a DNA β component for symptomatic infection. The DNA 1 molecule requires the helper begomovirus for movement in plants, but is capable of self-replication. We designed two abutting primer pairs (DNA101/DNA102 and UN101/UN102) to conserved sequences of DNA 1. This allowed polymerase chain reaction-mediated amplification of the full-length molecule from total nucleic acid extracts produced from various host plants from geographically distinct, worldwide locations. These primers are useful both as diagnostic probes and for producing full-length infectious clones for in planta studies.