光敏色素B基因(PHYB)启动子突变及与大白菜开花时间的关联分析

光敏色素B(phytochrome B,PHYB)是植物光周期依赖开花途径的关键调控因子之一。本研究对极端晚花和早花大白菜(Brassica rapa L.ssp.pekinensis)自交系06-247与He102的PHYB全基因组序列进行了生物信息学分析。结果表明,二者全长分别是5 986和5 528 bp,均与数据库中Chiifu-401的同源序列有不同程度的差异,表明二者均是大白菜PHYB基因的突变体,并命名为BraphyB1(GenBank登录号:KJ866947)和BraphyB2(GenBank登录号:KJ866948)。二者之间有476个插入/缺失(Inserts/Deletions,InDels)和57个SNPs。启动子区有473个InDels和31个SNPs、编码区有3个InDels和26个SNPs。InDels主要体现为phyB2在启动子区、5'非翻译区和编码区分别缺失445、18和3 bp。phyB2启动子缺失的445bp中包含两个TATABOX4和两处成髓细胞血症(myeloblastosis,MYB)类转录因子结合基序,一处GA响应元件。开发了鉴定phyB1/phyB2中445 bp InDel突变的共显性标记phyB1-762/phyB2-317,用该标记检测了以06-247与He102为亲本杂交构建的F2群体,利用F2代群体植株对抽薹开花时间与标记进行了关联分析,结果表明,phyB1-762/phyB2-317分别与晚花/早花表型显著关联(P0.05)。荧光定量RT-PCR显示,PHYB基因在He102各个发育阶段的表达水平均显著低于06-247(P0.01)。上述结果表明,大白菜PHYB基因启动子InDel是造成PHYB基因表达差异和开花期改变的主要原因之一。本研究为系统研究PHYB基因在转录、翻译、蛋白结构等水平突变对大白菜开花时间影响的分子机制提供了线索。     

基于BSA和SLAF-Seq技术对大豆主茎节数QTL精细定位

为了加速大豆主茎节数候选基因的的克隆和功能验证,并为大豆主茎节数分子标记辅助育种提供分子基础,本研究通过高通量测序检测与大豆主茎节数相关数量性状区间(QTL),结合双亲重测序信息开发QTL区间InDel分子标记,实现了大豆主茎节数相关主效QTL区间的精细定位。本研究以少主茎节数C025材料为母本,多主茎节数中119为父本杂交衍生的重组自交系(RIL)102个株系为试验材料,取自交系中极端少主茎节数30株和极端多主茎节数30株,构建两个极端混池,利用传统分群分析法(BSA)和全基因组特异性位点扩增片段测序手段(SLAF-Seq)相结合的方法在4号染色体检测到与大豆主茎节数相关的5个QTL。为了进一步缩小QTL区间,依据双亲材料的高通量重测序信息,获取QTL区间的插入缺失位点(InDel)信息,并开发InDel标记。首先利用InDel标记在F₂群体进行基因型分析,结果主效位点落在第3个QTL区间。其次在主效区间开发8个共显性InDel标记,结合RIL群体全部株系进行表型鉴定,最终获得9个交换单株,将主效区间分为6种交换类型,结合表型分析最终将大豆主茎节数位点精细定位到InDel标记Chr04-38和Chr04-46之间,其区间只有171.9 kb,包含候选基因6个,实现了大豆主茎节数的精细定位。本研究通过高通量测序与极端混池相结合的方法可以高效快速地检测与大豆主茎节数相关区间,并结合双亲重测序信息开发关联区间InDel分子标记,精细定位大豆主茎节数。本研究开发的Indel标记Chr04-38和Chr04-46与大豆主茎节数紧密连锁,有利于后期大豆主茎节数分子标记辅助育种。     

寒兰株系间遗传多样性和亲缘关系的SSR分子标记分析

本研究筛选了从春兰和莲瓣兰中开发的7个SSR位点的引物,用于37个寒兰株系的遗传多样性和亲缘关系研究。研究结果表明,7个SSR位点的引物在寒兰中均具有非常好的通用性,而且每个位点在不同的株系中具有特有等位基因,特别是位点CSSR05、CSSR11和195。株系具有的特有等位基因可以用于株系鉴别。7个位点在寒兰株系间具有...     

基于简化基因组测序技术的甘薯HRM分子标记开发及其应用

目前甘薯中针对SNP位点的分子标记开发及应用的报道较少。为开发甘薯特异SNP分子标记,本研究利用简化基因组测序技术对甘薯种质材料进行测序,筛选稳定的SNP位点,将其开发为基于HRM技术的分子标记,并在甘薯种质材料中进行验证。通过对23个甘薯种质材料简化基因组测序数据的分析,共发现835 756个SNP多态性位点,筛选其中的3 650个含有SNP多态性位点的高质量测序片段,成功设计了134对引物;初步验证发现,22对(16.42%)引物没有扩增产物,15对(11.19%)引物扩增产物2个以上,36对(26.87%)引物的扩增产物没有差异。61对(45.52%)引物在8个样本中的扩增特异性好,而且样本之间有差异。最后筛选34对扩增多态性丰富的引物对52份甘薯种质材料进行扫描,发现材料间多态性介于27.27%~90.91%之间,平均多态性达到59.35%。聚类分析表明,参试52份甘薯种质材料之间的差异较小,多数材料与骨干亲本南瑞苕、徐薯18之间关系较近,国外引进甘薯材料和地方品种差异较大。建议在今后甘薯育种中尽量选用地方品种和国外甘薯品种,从而更好地拓宽甘薯遗传背景。本研究初步建立了基于简化基因组技术和HRM技术开发甘薯SNP分子标记的新思路,为今后甘薯SNP分子标记开发提供了参考。同时本研究开发的甘薯分子标记,丰富了甘薯分子标记类型,为甘薯研究者开展快速的分子标记研究提供了支持。     

绵羊皮肤源EST-SSR标记与羊毛性状的关联性分析

为了筛选与绵羊(Ovis aries)毛性状基因座连锁更强的分子标记,本研究选择9个多态性丰富的绵羊皮肤源EST-SSR位点,检测了德国美利奴羊与中国美利奴羊级进杂交F2代群体的遗传多态性,并分析了标记与毛性状间的关联性.分析结果显示,所选的9个EST-SSR位点在供试群体中共检测到39个等位基因,多态性丰富,其中的6个位点基因型间存在显著差异;其中,与自然长度关联的位点有5个,与伸直长度关联的位点有4个,与纤维直径关联的位点有3个.6个EST-SSR位点基因型间存在显著差异;关联性标记对羊毛性状相关效应的F检验结果表明:33176918位点和33176988位点分别对羊毛自然长度、纤维平均直径具有显著的遗传效应(P＜0.05).研究结果提示,与羊毛性状相关的6个位点中,33176918号位点对羊毛的自然长度有较大的标记效应,该位点的CD基因型是羊毛自然长度的有利基因型;33176988号位点对羊毛的纤维直径有显著地标记效应,该位点的BC基因型是纤维直径的有利基因型.这两个位点可能与控制毛形状的基因座紧密连锁.     

蛋鸽雌激素受体基因多态性与产蛋量关联分析

雌激素受体(estrogen receptor,ESR)是核受体超家族中的一种配体依赖的转录因子,大量研究显示,ESR基因与动物的繁殖活动密切相关.为了分析ESR基因单核苷酸多态性(single nucleotide polymorphism,SNPs)及其与蛋鸽(Columba livia)产蛋量的关系,本研究以泰平王鸽(C.livia)215个样本为研究对象,采用第一代测序法对ESR基因部分片段进行SNPs筛选.在所测的3个片段中共发现了10个突变位点,分别为ESRα基因的T129416C,ESRβ基因的T365C、A388G、C389A、A429G、G451A、G577A、T672C、A923C和T17167C,其中有3个突变位于外显子上,但只有位点G577A导致了氨基酸的改变,由氨基酸异亮氨酸(isoleucine,Ile)转变为缬氨酸(valine,Val).对10个位点进行了Hardy-Weinberg平衡状态检验,结果显示,所有位点均处于平衡状态.10个位点的连锁不平衡分析显示,位点S2与位点S4、S9为强连锁不平衡(D'≈1,r2＞0.8),位点S4、S9可以完全代表S2.多态位点与产蛋量关联分析显示,位点S8与产蛋量呈极显著相关,其中杂合子AB型的平均产蛋量极显著的高于纯合子AA型(P＜0.01);而纯合子BB型的平均产蛋量低于AB型,高于AA型,但差异不显著(P＞0.05).本研究表明,ESRβ基因中位点S8可作为蛋鸽产蛋量性状选育的候选分子标记位点,为蛋鸽的分子标记辅助育种提供资料.     

利用RAPD与SSR技术进行野生大豆种群内分化的研究

利用RAPD和SSR技术对25°N野生大豆种群16个样本进行分子标记,从150多个RAPD引物中筛选出具有多态性的引物20个,共扩增出146个标记位点,其中具有多态性的有60个,占总位点的40.8%,平均遗传距离为0.1536,杂合度为0.3248.从60对SSR引物中筛选出14对具有多态性的引物,平均遗传距离为0.2209,杂合度为0.6961.聚类分析可将25°N野生大豆16个样本分成5类,表明种群内存在大量的遗传变异.为研究野生大豆种群内及种群间的遗传多态性探索了有效方法,并提出野生大豆核心种质资源的保存及取样对策.     

空间诱变玉米自交系齐319的SSR标记变异分析

玉米自交系齐319经"实践八号"育种卫星搭载后,发现两个籽粒诱变系SP-M1和SP-M2,由硬粒变为中齿和马齿。为解析籽粒变化原因,本文选用分布在玉米10条染色体上的385对SSR标记对两个变异株系和齐319进行变异分析。研究表明,SP-M1和SP-M2诱变系与齐319遗传相似系数分别为0.715和0.682,说明诱变系与野生材料之间存在遗传差异;中齿诱变系(SP-M1)与齐319之间检测到40个SSR变异位点,变异频率为10.93%,其中32个位点表现为扩增片段长度变异,8个位点表现为扩增片段数目变化;马齿诱变系(SP-M2)与齐319之间检测到55个SSR变异位点,变异频率为15.03%,其中47个位点表现为扩增片段长度变异,8个位点表现为扩增片段数目变化。不同染色体上位点变异频率差异较大,变异位点表现出成簇分布的特点,主要分布在第1、2、3、6、7、9、10条染色体上。本研究为探讨玉米空间诱变机制提供参考。     

大白菜简单序列重复(SSR)和插入/缺失(InDel)标记的开发及通用性分析

为探讨大白菜基因组序列中SSR位点的分布规律并开发SSR引物,利用SSRHunter软件对大白菜A10(16899818～17299817)的DNA序列进行简单序列重复(SSR)位点查找,共得到394个SSRs,平均每1.02kb出现1个SSR。二核苷酸和三核苷酸重复是最主要的SSR类型,分别占79.44%和18.78%。为了提高SSR标记开发的准确性和通用性,对检索得到的含SSR位点的序列进行了同源比对,选取符合条件的15条SSR序列并设计引物;依据Blast过程中发现的在SSR位点不存在差异而在其侧翼序列中存在插入/缺失(InDel)差异的序列,设计了19条InDel引物。用34对SSR及InDel引物在6个大白菜(Brassica rapassp.pekinesis)材料中进行多态性研究,发现28对引物能扩增出理想的PCR产物,有效扩增率为82.35%,其中27对引物具有多态性,多态性比率为79.41%。为验证SSR引物的真实性,随机对4对SSR引物的部分白菜扩增片段进行了测序,发现100%的片段具有相应的SSR位点。28对SSR和InDel引物在甘蓝(B.oleracea)、油菜(B.napus)和萝卜(Raphanus sativus)品种的有效扩增率分别为85.71%、100%和77.78%,说明新开发的SSR和InDel标记具有较好的多态性和通用性。利用6对引物分析了48份十字花科种质的遗传多样性,结果表明48份材料被明显地区分成白菜和甘蓝组、萝卜组、油菜组3大类群,与传统分类一致。大白菜SSR和InDel标记的开发对于十字花科种质亲缘关系及遗传多样性分析具有重要的应用价值。     

南阳牛Gli3基因多态性及其与生长性状的相关性

Gli3基因是Gli-Kruppel家族中的一员,其参与的Sonic hedgehog信号通路在动物生长发育,特别是早期生长发育过程中有重要作用.利用PCR-SSCP和DNA测序技术研究了187头纯种南阳牛Gli3基因编码区4个位点(E4、E5、E6和E7)的多态性,发现E4和E5位点存在SSCP多态.对不同SSCP带型的对应片段进行了测序分析,共发现3个新的SNP多态位点(T123600C、T123696C和A128688G),其中第123600和第123696两个位点处于连锁状态.E5位点处于Hardy-Weinberg平衡状态,E4位点处于非平衡状态.不同基因型与生长发育性状的相关性分析显示,E4位点上,CC基因型个体的六月龄体重、胸围、坐骨端宽、日增重指标显著大于TT基因型个体(P<0.05),该位点有可能作为南阳牛早期生长性状标记辅助选择的标记之一.     

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.     

Predicting the Classes and Distribution of Salt-Affected Soils in Northwest Libya

Sodicity and salinity can adversely affect soil structure and are common constraints to plant growth in arid regions. Current remote sensing techniques cannot distinguish between the various classes of salt-affected soils. Field and laboratory measurements of salt-affected soils are time-consuming and expensive. Mapping of the salt-affected soils can be used in soil conservation planning to identify regions with different degrees of limitations. There is a need to use existing field and laboratory measurements to create maps of classes of salt-affected soils. The objectives of this study are to classify salt-affected soils, use existing field data to interpolate and validate geospatial predictions of the classes of salt-affected soils using Geographic Information Systems (GIS), and create maps showing the different classes and distribution of salt-affected soils. The classification framework for salt-affected soils is based on electrical conductivity (ECe), soil pH and the sodium adsorption ratio (SAR), and provides four degrees of limitations to salt-affected soils: slight (normal soils), moderate (saline soils), severe (sodic soils), and extreme (saline-sodic soils). Spatial interpolation of the field data from northwestern Libya was verified by cross-validation, and maps of the salt-affected soils in the region were created. The majority of soils in this region of Libya are normal (slight degree of limitation). Twenty percent of the topsoil is saline-sodic (extreme degree of limitation). Land use recommendations and rehabilitation strategies can be developed from such maps of salt-affected soil classes. The methodology followed in this study can be applied to other arid regions around the world, particularly in developing countries where budgetary constraints limit detailed field and laboratory measurements of sodicity and salinity.     

黑龙江农田土壤壬基酚及其短链聚氧乙烯醚残留调查

对黑龙江土壤样品中的壬基酚(NP)、壬基酚一氧乙烯醚(NP1EO)和壬基酚二氧乙烯醚(NP2EO)的残留状况开展研究。结果表明,NP、NP1EO和NP2EO均有不同程度的检出,检出率在68.46%~95.38%之间;NP、NP1EO和NP2EO的浓度分别为ND~156.8μg kg-1、ND~15.70μg kg-1和ND~13.63μg kg-1,均值分别为22.02μg kg-1、4.04μg kg-1和2.15μg kg-1;进一步分析,已检出的样品中,NP在0~10μg kg-1的比重最大,NP1EO在4~6μg kg-1比重最大,NP2EO在1~2μg kg-1比重最大。NP的残留水平高于NP1EO和NP2EO,说明NP相对较难降解,更易在土壤中累积。     

Phosphorous accumulation and distribution of two ecotypes of Pilea sinofasciata grown in phosphorous-enriched soils

Water eutrophication and poultry manure require an efficacious remediation technology to decrease phosphorous (P) concentration. In this study, the P phytoremediation potential of a mining (ME) and nonmining ecotype (NME) of Pilea sinofasciata were analyzed by pot experiments employing different concentrations of P treatments (CK, P100, P200, P400, P800 and P1600) in tested soil. Growth, P accumulation and P-relevant enzyme activities of P. sinofasciata in ME and NME were assessed after 10 weeks’ plantation. The results showed that there was no significant difference between the shoot biomass of the ME and NME. Higher P concentration and accumulation were observed in the ME than NME for all treatments. Stem P concentration of the ME were 117%, 189%, 152%, 153%, 140% and 139% higher than the NME, and leaf P concentration were 122%, 146%, 138%, 144%, 137% and 113%. Maximum P uptake and phytoextraction was observed at P400 for the ME of P. sinofasciata. Elevated activities of acid phosphatase and phytase in roots were examined in P-enriched soil, and the ME presented higher activities compared with the NME. The results in this paper provide a theoretical basis for the use of ME of P. sinofasciata for phosphorus accumulators and suggest that ME of P. sinofasciata is a promising species for the excess P phytoextraction of P-enriched soils.     

有限性灌溉对设施草莓产量及水分利用效率的影响

在日光温室环境及盆栽条件下,通过与全灌(FI)比较,研究了亏缺灌溉(DI)及分根交替灌溉(PRD)对草莓产量和灌水利用效率(IWUE)的影响.试验从开花期开始到果实成熟期结束.共分5个处理,全灌(FI);分根灌溉PRD分2个处理水平,即PRD1和PRD2;限制灌溉DI也分两个处理水平,即D11和D12.与F1处理相比,DI1和DI2处理的叶水势和气孔导度明显偏低,单株叶面积、鲜果重、平均单果重也显著低于FI处理.但水分利用效率却比FI处理的高(分别高15.8%和29.3%);PRD1处理的叶水势与FI处理的相当.且高于其它处理,而气孔导度比FI,DI1和D12处理的明显降低,与PRD2处理的相近.PRD1处理的鲜果重、平均单果重与FI处理的相当,比DI1,DI2和PRD2处理的显著提高;与FI处理相比,DI1,DI2,PRD1和PRD2处理分别节水31.5%,50.0%,31.5%和50.0%,水分利用效率分别提高了15.8%,29.3%,44.4%和84.4%,从草莓产量和水分利用效率各指标可以看出,PRD1处理比DI1,DI2和PRD2处理表现出明显的优势,其综合效益最佳.     

Effects of residue coverage on the characteristics of soil carbon pools in orchards

The effects of organic waste coverage on the quality of soil carbon (C) pools in orchards remain unclear. In this study, we performed a 3-year experiment in an 8-year-old Fuji apple orchard to investigate the effects of inter-row weed growing/inner-row pruning residue coverage (PRC), weed coverage (WC), and inter-row weed growing/inner-row non-coverage (used as a control) on the contents of soil water, available nitrogen, phosphorus, and potassium, organic C components, glomalin-related soil protein (GRSP), soil microbial biomass C, and the number of microorganisms at different soil depths. PRC increased the percentage of readily oxidizable C (ROC), whereas WC improved the percentage of soil humin (HM). Thus, PRC enhanced SOC activity, whereas WC promoted SOC stability. Moreover, soil water, available nitrogen, phosphorus, and potassium contents increased in WC in 0–20-cm soil depths as compared to that in PRC, and the correlation between total GRSP (T-GRSP) and HM (r = 0.92) was higher than that between T-GRSP and ROC (r = 0.79). Therefore, T-GRSP is important in HM formation and maintaining stability of C pools. Overall, these results improve our understanding of the positive effect of organic waste coverage on soil internal C cycling and energy flow within an orchard ecosystem.     

溶解性有机质对镉在蔬菜中累积的影响

在赤红土中施用溶解性有机质（ＤＯＭ）——稻秆ＤＯＭ（稻秆发酵液中的有机质）后，生长在该土壤上的蔬菜体内镉含量，较不施稻秆ＤＯＭ的显著增加，加重了对蔬菜的危害程度     

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。     

Effects of nitrification inhibitors on denitrification of nitrate in soil

Summary The influence of 28 nitrification inhibitors on denitrification of nitrate in soil was studied by determining the effects of different amounts of each inhibitor on the amounts of nitrate lost and the amounts of nitrite, N₂O and N₂ produced when soil samples were incubated anaerobically after treatment with nitrate or with nitrate and mannitol. The inhibitors used included nitrapyrin (N-Serve), etridiazole (Dwell), potassium azide, 2-amino-4-chloro-6-methylpyrimidine (AM), sulfathiazole (ST), 4-amino-1,2,4-triazole(ATC),2,4-diamino-6-trichloromethyl-s-triazine (CL-1580), potassium ethylxanthate, guanylthiourea (ASU), 4-nitrobenzotrichloride, 4-mesylbenzotrichloride, sodium thiocarbonate (STC), phenylmercuric acetate (PMA), and dicyandiamide (DCD).Only one of the nitrification inhibitors studied (potassium azide) retarded denitrification when applied at the rate of 10 g g^–1 soil, and only two (potassium azide and 2,4-diamino-6-trichloromethyl-s-triazine) inhibited denitrification when applied at the rate of 50 g g^–1 soil. The other inhibitors either had no appreciable effect on denitrification, or enhanced denitrification, when applied at the rate of 10 or 50 g g^–1 soil, enhancement being most marked with 3-mercapto-1,2,4-triazole. Seven of the inhibitors (potassium azide, sulfathiazole, potassium ethylxanthate, sodium isopropylxanthate, 4-nitrobenzotrichloride, sodium thiocarbonate, and phenylmercuric acetate) retarded denitrification when applied at the rate of 50 g g^–1 soil to soil that had been amended with mannitol to promote microbial activity.Reports that nitrapyrin (N-Serve) and etridiazole (Dwell) inhibit denitrification when applied at rates as low as 0.5 g g^–1 soil could not be confirmed. No inhibition of denitrification was observed when these compounds were applied at the rate of 10 g g^–1 soil, and enhancement of denitrification was observed when they were applied at the rate of 50 or 100 g g^–1 soil.     

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.