柑橘黄龙病菌侵染九里香不同方法研究

采用不同的方法将柑橘黄龙病菌(Candidatus Liberibacter asiaticus)传染到九里香(Murraya paniculata),运用Nested-PCR技术确认九里香是否感染黄龙病菌.结果表明,采用汁液摩擦、注射和挤压法,接种2个月后不能在九里香植株体内检测到黄龙病菌;黄龙病阳性接穗红江橙(Ci...     

鉴定柑桔病毒病的三种指示植物秋季嫁接适宜温湿度初探

指示植物鉴定是柑桔病毒病鉴定的传统方法。由于其结果观察的直观性、鉴定结果的可靠性和能准确地反映病毒的生物学特性,指示植物鉴定目前仍在广泛应用。柑桔多采用嫁接接种病原,常用的嫁接接种法有双重芽接法和指示植物直接嫁接法。广西柑桔研究所通常采用双重芽接法,即在无病砧木上同时嫁接1个指示植物芽片和2个待检芽片（皮或枝段）。接种类似繁育柑桔苗木,     

山东几个苹果主产区三种潜隐病毒的鉴定

对山东省6个生产苹果县(市)的果园部分主栽品种和近年来引进的北斗、红富士等苹果新品种及M26等矮化砧木中主要潜隐病毒种类进行了木本指示植物鉴定。木本指示植物是苏俄苹果(R12740-7A)、光辉(Radient)、司派227(SPY227)和弗吉尼亚小苹果(Virginia crab)。检测方法采取双重芽接法,即被检品种的接芽嫁接在检测圃内实生海棠砧木的基部,约离地面5cm,其上3～5cm处嫁接指示植物的芽片。嫁接1周后检查成活率,未嫁接成活的再行补接;10天后剪除接芽上砧木部分,待被检芽生长到10cm以上时,剪除顶尖促使指示植物芽的生长,观察症状的出现和带毒率。…     

柑桔黄龙病病株感染衰退病毒情况的鉴定

在我国南部柑桔产区,黄龙病是一种十分严重的病害。此病在我国西南的部分地区亦有发生。此病的病原,陈其爆(1943)通过调查和试验,认为最大的可能是病毒。林孔湘(1956)进一步试验证明病害可以通过嫁接传播,明确了病毒病原。福建的嫁接传病试验亦证实了这一点。 从国内外柑桔病毒病发生情况的分析对比中,以及根据利用指示植物鉴定证明黄龙病树受衰退病毒(tristeza)感染,曾经有人认为黄龙病的病原就是衰退病毒或可能是衰退病     

迟熟蕉柑茎尖嫁接及其病毒鉴定

采用田间结果树上的芽作为接穗进行茎尖嫁接,最终获得7株茎尖嫁接苗。对所获得的茎尖嫁接苗,应用指示植物腊斯克枳橙、墨西哥来檬和伊特洛格香橼亚利桑那861-S-1分别鉴定柑橘碎叶病、衰退病和裂皮病,用PCR检测技术鉴定柑桔黄龙病。结果表明：1、3、4、5、6号迟熟蕉柑单株不带碎叶病、衰退病、裂皮病和黄龙病,可用作繁殖无病毒苗木的备选材料。     

苹果常见病毒病害田间指示植物检测试验小结

“农业部与山东省联建山东国家级果树无病毒苗木繁育基地烟台脱毒检测中心”1991年开始由农业部牵头投建,省果茶站代管。5年来,我所先后由联邦德国、中国果树所(兴城)等处引进一批果树病毒病害检测用木本指示植物与草本指示植物及一批果树无病毒材料。为了验证这些引进指示植物及无病毒材料的可靠性,试验总结规范化的指示植物田间检测技术规程及常现应用的可行性,择样进行了本试验。现将试验情况简结如下。     

苹果潜隐病毒的温室鉴定

目前,已知的苹果潜隐病毒都不能或难于接种到草本植物上。因此,病毒种类的鉴定,主要是利用木本指示植物二重嫁接法在田间进行。虽然木本指示植物田间鉴定法可以鉴定所有已知的苹果潜隐病毒,但其缺点是所需时间长,若将所有病毒种类鉴定清楚,则需要2～4     

柑桔黄龙病菌的重组酶聚合酶扩增(RPA)检测

为了建立黄龙病菌的重组酶聚合酶扩增(Recombinase Polymerase Amplification, RPA)快速检测体系,丰富柑桔黄龙病的高效简便检测方法,以黄龙病菌株psy62全基因组中的单拷贝tufB-secE-nusG-rplKAJL-rpoB基因簇序列和5拷贝核糖核苷酸还原酶β亚基基因(nrdB)序列设计并筛选特异性引物,经灵敏度比较和检测特异性验证,建立该病的RPA检测体系,并经PCR、RPA和实时荧光PCR对87份柑桔样品的黄龙病菌进行对比检测,验证RPA检测体系的适用性。结果表明,建立的黄龙病菌RPA检测方法能特异性检测柑桔黄龙病;灵敏度与实时荧光PCR相当,是PCR的100倍;扩增过程只需20 min,大幅提高检测效率;对田间疑似柑桔黄龙病的送检样品检测结果与实时荧光PCR一致,比PCR的检出率高。     

田间耐柑橘黄龙病橘类品种材料的评价研究

研究对广西田间疑似耐柑橘黄龙病橘类品种材料进行了收集,共收集了82份橘类品种材料,其中沙糖橘28份、椪柑46份、桂橘1号5份、南丰蜜橘1份及蜜广橘2份,对收集的疑似耐病材料进行嫁接和保存,并建立国内首个耐柑橘黄龙病橘类品种材料保存圃。通过高接染毒法对疑似耐黄龙病橘类柑橘品种材料进行鉴定,结合田间症状观察诊断和定性/定量PCR检测结果,初步筛选出13份对黄龙病有一定耐性的橘类品种材料,为柑橘的抗病育种提供依据。     

柑桔指示植物鉴定接种方法改进研究

应用传统接种方法进行指示植物鉴定时,每株苗只能鉴定一种柑桔病害;但应用改进接种方法,将分别用来鉴定柑桔碎叶病、衰退病、裂皮病的腊斯克枳橙、墨西哥来檬、861-S-1香橼等3种指示植物的芽同时嫁接接种在一株枳砧上,就可以用一株苗同时鉴定3种柑桔病害.本试验以已知带病的柑桔品种作为被鉴定试材,应用该改进接种方法和传统接种方法分别对其进行鉴定,结果显示,该改进接种方法和传统接种方法的鉴定结果一致,也和被鉴定材料已知带病情况一致,且该改进接种方法和传统接种方法的指示植物病情指数之间并无显著性差异.     

杏鲍菇枯萎病菌PCR检测方法的建立与初步应用

以杏鲍菇枯萎病菌侧耳泛菌(Pantoea pleuroti)为试材,采用PCR引物设计的方法,通过分析P.pleuroti基因组的测序结果,设计合成并筛选出可检测P.pleuroti的引物,并研究其检测效果,以期建立P.pleuroti的高效PCR检测体系,并为科学防控杏鲍菇枯萎病提供分子基础。结果表明:K3143F/R和K37F/R 2对引物特异性强,仅P.pleuroti基因组DNA作为模板时,PCR扩增产物分别呈现1条660 bp和1条666 bp的特异性条带,15株Pantoea属细菌和3株食用菌常见病原菌的基因组DNA及阴性对照作为模板扩增产物均无条带;基于这2对引物建立的检测体系均不受杏鲍菇组织液的干扰,灵敏度高,可检测出最低3.6 pg·μL^-1的P.pleuroti基因组DNA;应用这2种体系对接种P.pleuroti 48 h后的杏鲍菇样品进行了检测,最低可检测出子实体内100 cfu的杏鲍菇枯萎病菌;此外,整体上,引物K3143F/R比K37F/R的检测效率更高。     

安祖花细菌性疫病的Nested-PCR检测

根据基因库中已发表的安祖花细菌性疫病致病菌粉黛黄单胞杆菌（Xanthomonas campestris pv.dieffenbachiae）基因序列设计引物XcF1/XcR1、XcF2/XcR2,通过扩增条件优化,建立了安祖花细菌性疫病的Nested-PCR检测方法,可以直接从感染细菌性疫病的安祖花组织DNA中扩增出1...     

Impact of HBV DNA level in serum on the cloning efficiency of HBV full length genome

AIM: To compare the cloning efficacy of full-length HBV genome amplified by single fragment PCR and two fragment PCR for choosing the suitable method for full-length HBV genome cloning. METHODS: To amplify the full-length HBV genome from 85 sera sample of HBV patients, single fragment PCR and two fragment PCR were conducted. The products were cloned into the vector and sequenced after identified with double enzyme digestion. At the same time, the titers of 85 samples were detected by real-time PCR. RESULTS: Compared with two fragment PCR, single fragment PCR requested higher level of sera HBV DNA for successful amplification of full-length HBV genome, and the efficacy of single fragment PCR was lower than that of two fragments PCR (P<0.05). The mutation ratio of single fragment PCR was 1.13 bp/kb, and the sensitivity of single fragment PCR was 102 original templates. The efficacy of amplification was 80% if the amounts of template exceed 103, but the efficacy was low under this value. CONCLUSION: The efficacy of amplification is affected by the level of sera HBV DNA. The titers of sera HBV DNA are the proof for choosing a suitable PCR method. If the level of sera HBV DNA was more than 106 copies/L, single fragment PCR will be suitable. If the level of sera HBV DNA was less than 106 copies/L, two fragments PCR will be better.     

应用基因芯片检测中国野生葡萄抗白粉病基因表达

 本文研究了利用Affymetrix的欧洲葡萄基因表达芯片检测中国野生葡萄基因表达的可行性,并检测了葡萄白粉菌(Uncinular necator)人工接种诱导后48 h后的基因表达情况。结果表明,该芯片可以在感病材料中检测出表达的基因11 906个,抗病材料中检测到表达的基因11 839个,在二者中同时检出表达的基因11 839个,能够检出的基因数占该芯片基因探针组总数的71.3%,因此用欧洲葡萄基因芯片检测中国野生葡萄中基因表达水平是完全可行的;并利用此芯片检测出在人工接种诱导后,抗病材料中和感病材料相比表达上调的基因共1 920个,占检测出基因总数的16.21%;表达下调的基因数1 760个,占检测出基因总数的14.87%;表达上调和下调的幅度(signal log ratio)都在1~7倍之间。     

甜菜根际土壤AM真菌分离与分子鉴定

为了揭示丛枝菌根（arbuscular mycorrhiza,AM）真菌与甜菜的共生关系,收集甜菜根系及根际土壤,采用湿筛倾析-蔗糖离心法分离甜菜根围土壤AM真菌孢子。利用形态学和分子生物学方法对分离得到的AM真菌抱子进行分类鉴定,并应用Nested—PCR技术检测甜菜根际土壤AM真菌侵染甜菜根系情况。依据AM真菌孢子形态特征及25SrDNA D1／D2区域序列分析,鉴定出甜菜根围土壤中具有摩西球囊霉（Glomus mosseae）,并且应用Nested—PCR技术从甜菜根内检测到了G．mosseae,表明G．mosseae侵染甜菜根系。     

用RT-PCR检测PRSV在番木瓜体内的运转

以抗病突变体岭抗1号与穗中红番木瓜为试材,人工接种PRSV-Ys株系,应用RT-PCR技术检测PRSV在体内的运转.结果表明,感病品种穗中红接种后24 h,即可在接种叶的未接种部位检出病毒,第10 d在接种植株的各部位均可检出病毒;而在岭抗1号植株上,接种后直至第10 d,仍未能在其他部位检测出病毒,表明岭抗1号具有阻碍病毒运转的能力.     

中国野生毛葡萄芪合酶基因抗白粉病功能分析

中国野生毛葡萄（Vitis quinquangularis）‘丹凤–2’白藜芦醇含量高，并抗白粉病。以此为材料，利用同源序列克隆技术获得两个芪合酶基因 VqSTS12 和 VqSTS25。以欧洲葡萄‘无核白’分生愈伤组织为受体材料，通过农杆菌介导法进行遗传转化，分别获得 7 个 VqSTS12 和 5 个 VqSTS25‘无核白’葡萄转基因植株。实时定量 PCR 分析发现，与野生型植株相比，转基因植株中芪合酶基因 STS 与下游白藜芦醇糖基转移酶基因 RSGT 表达量升高，与 STS 有底物竞争关系的查尔酮合酶基因 CHS 表达量降低。STS 的表达产物中主要的芪类物质反式云杉新苷含量增加。选择芪类物质含量高的转基因植株接种白粉菌发现，转基因植株对白粉菌的敏感性降低，发病较晚，病情较轻，在转基因植株上部分孢子萌发受抑制，菌丝发育迟缓。转基因植株中 VqSTS12 和 VqSTS25 受白粉菌诱导表达：在接菌后 1 ~ 3 d 内表达量显著升高，随后略有下降，在 6 ~ 7 d 表达量再次上升。同时高效液相色谱（HPLC）检测到 STS 的表达产物白藜芦醇、云杉新苷、葡萄素与紫檀芪含量增加。研究结果表明‘丹凤–2’毛葡萄拥有的 VqSTS12 和VqSTS25 及其表达的芪类物质具有抑制白粉菌的作用。     

Detection of miR-122 expression in serum by Taqman probe real-time fluorescence quantitative PCR and its clinical significance

AIM: To detect the serum level of miR-122 expression by the technique of Taqman probe real-time fluorescence quantitative PCR for identifying its clinical significance. METHODS: The stem-loop RT primer, PCR primer and Taqman probe of miR-122 and U6 snRNA were designed. The expression of miR-122 in the serum samples of 27 cases of preoperative hepatocellular carcinoma (HCC), 15 cases of hepatitis B, 15 cases of hepatitis C, 15 cases of healthy control (HC), 11 cases of postoperative hepatocellular carcinoma (PHCC) and 10 cases of recurrence after postoperative hepatocellular carcinoma was detected by Taqman probe real-time fluorescence quantitative PCR. U6 snRNA was used as the internal control. RESULTS: The results showed that the method of Taqman probe real-time fluorescence quantitative PCR could detect the amplification signal of serum miR-122. The expression level of serum miR-122 in the patients with HCC, hepatitis B, hepatitis C and recurrence was higher than that in HC and the patients with PHCC. Meanwhile, the serum level of miR-122 in the patients with hepatitis C was higher than that in the patients with HCC, hepatitis B and recurrence. However, no difference of miR-122 expression level among HCC, hepatitis B and recurrent patients was observed. The miR-122 level was lower in PHCC patients than that in HCC and recurrent patients. In hepatitis B virus surface antigen (HBsAg) and/or hepatitis B virus e antigen (HBeAg) positive patients, the miR-122 level was higher than that in the negative ones. The miR-122 level in hepatitis C antibody (HCV-Ab) positive patients was raised compared with the negative ones. The serum level of alanine aminotransferase (ALT) was positively correlated with the serum level of miR-122 (r=0.34, P<0.05). The miR-122 expression level in the patients with serum AFP≥400 μg/L was higher than that in the patients with serum AFP<400 μg/L. CONCLUSION: The method of Taqman probe real-time fluorescence quantitative PCR can detect the serum level of miR-122 expression. Serum miR-122 might be used as a new biomarker of liver diseases, especially in the early diagnosis of primary hepatocellular carcinoma, the curative effect of surgical operation and the prognosis.     

Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments

ABSTRACT: BACKGROUND: Multiplex PCR has been successfully applied in many areas since it was first reported in 1988; however, it suffers from poor universality. RESULTS: A novel method called Universal Multiplex PCR (UM-PCR) was created, which simultaneously amplifies multiple target fragments from genomic DNA. The method has two steps. First, the universal adapter-F and universal adapter-R are connected to the forward primers and the reverse primers, respectively. Hairpin structures and cross dimers of five pairs of adapter-primers are detected. Second, UM-PCR amplification is implemented using a novel PCR procedure termed "Two Rounds Mode" (three and 28-32 cycles). The first round (the first three cycles) is named the "One by One Annealing Round". The second round (28-32 cycles) combines annealing with extension. In the first two cycles of the first round, primers only amplify the specific templates; there are no templates for the universal adapters. The templates of universal adapters begin to be synthesized from the second cycle of the first round, and universal adapters and primers commence full amplification from the third cycle of the first round. CONCLUSIONS: UM-PCR greatly improves the universality of multiplex PCR. UM-PCR could rapidly detect the genetic purity of maize seeds. In addition, it could be applied in other areas, such as analysis of polymorphisms, quantitative assays and identifications of species.