复合PCR技术在混合感染检测中的应用

用传统的方法检测传染性疾病一次只能检测一种病原体,若为混合感染时,检测繁琐、费时且成本高,并且常常造成漏检.为了有效地解决上述问题,本文利用复合PCR分子诊断技术,建立了一套对由沙眼衣原体和解脲支原体造成的混合感染进行检测的方法.结果表明,该方法高效快速,可以开发成诊断试剂盒应用于混合感染的检测中.     

利用多重PCR探究男性尖锐湿疣与HPV感染之间的相关性

本文的主要目的是建立一种针对人乳头瘤病毒(Human Papillomavirus, HPV)检测的多重PCR方法,并利用这种方法探究男性尖锐湿疣与HPV感染之间的相关性.首先建立一种检测HPV的多重PCR方法,然后运用该方法对156例男性尖锐湿疣标本的HPV3种低危(HPV6,11,42)和6种高危(HPV16,18,33,52,56,58)亚型进行检测,并与反向膜杂交检测结果进行比较.结果156例标本中阳性标本数69例,占总标本数的44.23%,其中单一感染标本数48例,占69.57%,混合感染标本数     

五黑乌骨鸡与黄山黑鸡性能比较及微卫星多样性分析

为了解安徽地方黑鸡品种生长发育、繁殖性能和遗传距离,比较分析了五黑乌骨鸡和黄山黑鸡外貌特征、体尺发育、蛋品质和繁殖性能,以及微卫星标记在2个群体中的遗传多样性。结果表明,五黑乌骨鸡体质量、体长、骨盆宽和胫长均显著高于黄山黑鸡（P < 0.05）,胸宽和胸深显著低于黄山黑鸡（P < 0.05）,龙骨长和胫围在2品种间差异无统计学意义（P > 0.05）。五黑乌骨鸡蛋重、蛋壳厚度、蛋壳强度和蛋黄比显著高于黄山黑鸡（P < 0.05）。2品种间蛋壳厚度无统计学意义（P > 0.05）。五黑乌骨鸡的绿壳蛋率为71.2%,黄山黑鸡蛋壳粉色。MCW0034等9个微卫星标记在2个群体中共检测到140个等位基因,不同座位上的等位基因大小在56~341 bp之间;平均多态信息含量为0.760 4,平均有效等位基因数为6.428 7,平均基因杂合度为0.785 0。五黑乌骨鸡蛋品质高于黄山黑鸡,可能与体重和体尺指标有关。微卫星标记的研究结果为我省地方黑鸡品种的种质特性研究提供了基础数据,为地方鸡种资源合理保护和利用提供了理论依据。     

Genetic analysis and fine mapping of a lax mutant in rice

We have analyzed a lax mutant that exhibits altered panicle architecture in rice.The primary and secondary rachis-branches are normally initiated and each branch ends in a terminal spikelet,but all the lateral spikelets are absent and the terminal spikelet displays variegated structures in the mutant.An F2 population from the cross between the lax mutant and a japonica variety,W11,was constructed and analyzed.Using microsatellite and CAPS markers,the lax locus was mapped on the long arm of chromosome 1,co-segregated with a CAPS marker,LZ1,within an interval of 0.28 cM between a CAPS marker,HB2,and a microsatellite marker,MRG4389.RT-PCR analysis revealed that the expressions of the rice B-function MADS-box genes OsMADS2,OsMADS4,OsMADS16 and OsMADS3 were significantly reduced,whereas the expression of the rice A-function gene RAPIA was not altered.     

玫瑰冠鸡资源群的微卫星多态性分析

利用8个微卫星DNA标记分析了玫瑰冠鸡(50只)及其杂交鸡(40只)的群体遗传变异。计算了各群体在各位点上的等位基因频率,并据此计算出各群体的平均遗传杂合度、多态信息含量和有效等位基因数。结果表明:2个鸡群在8个微卫星座位上的基因频率存在明显的差异。所选的8个微卫星座位均为高度多态,可作为有效的遗传标记用于鸡群体遗传多样性的分析。     

我国药用价值地方鸡种的遗传多样性研究

通过选用30个多态性较好的微卫星标记,检测了9个药用价值地方鸡品种:郧阳白羽乌鸡、乌蒙乌骨鸡、盐津乌骨鸡、腾冲雪鸡、余干乌骨鸡、兴文乌骨鸡、石棉草科鸡、沐川乌骨鸡、泸宁鸡的遗传多样性。利用等位基因频率计算各群体平均遗传杂合度(H)、多态信息含量(PIC)和群体间的遗传距离(Ds)。结果表明:30个微卫星位点中有24个微卫星位点在9个鸡群体中PIC均为高度多态,可作为有效遗传标记用于各鸡品种的遗传多样性和系统发生关系的分析。在9个品种中,杂合度最低的是郧阳白羽乌鸡,为0.640;杂合度最高的是腾冲雪鸡,为0.690。因此药用价值地方品种鸡的H都偏高,据分析可能是这些药用价值地方品种鸡都产于交通闭塞的山区,形成了家禽品种的多种多型,而且杂合度的高低与PIC值的大小体现了较高的一致性;对Ds的计算表明:体格较大的泸宁鸡与萁它品种距离较远;用UPGMA法进行聚类分析,结果9个鸡品种被聚为3类:郧阳白羽乌鸡、沐川乌骨鸡、盐津乌骨鸡和腾冲雪鸡聚为一类;余干乌骨鸡、兴文乌骨鸡、乌蒙乌骨鸡和石棉草科鸡聚为一类;泸宁鸡独自聚为一类。这与几个鸡品种的来源、分化、选育历史及地理分布是一致的。     

多重PCR法快速鉴定转基因小麦植株及后代

根据转基因植物中常用的报告基因uidA、选择基因bar的序列,设计合成两对不同的引物,建立了在转基因小麦材料分子确证中,应用一次PCR反应同时扩增检测两种或多种外源基因的多重PCR方法．通过对质粒pAHC25以及30个转基因小麦样品进行PCR扩增分析,比较多重PCR的检测结果与单基因的PCR扩增结果,发现两者结果完全一致．     

Characterization and mapping of a white panicle mutant gene in rice

A spontaneous white panicle mutant was found from the F6 progenies of an indicajaponica cross.The mutant exhibits white stripes on its basal leaves while the panicles,rachis and pedicel are milky white colored at flowering stage.Genetic analysis in an F2 population from the cross of Zhi7/white panicle mutant indicates that the white panicle phenotype is controlled by a single recessive nuclear gene,tentatively termed as wp(t).Using microsatellite markers,the wp(t) gene was anchored between the markers of SSR101 and SSR63.9 with a map distance of 2.3 and 0.8cM,respectively,and co-segregated with the marker of SSR17 on rice chromosome 1.     

多重PCR检测病死鸡中沙门氏菌方法的研究

为了建立能够同时检测病死鸡样品中的沙门氏菌、鸡白痢沙门氏菌和肠炎沙门氏菌的多重PCR 方法.采用煮沸法提取 DNA 做为模板,选择分别针对沙门氏菌属、鸡白痢沙门氏菌和肠炎沙门氏菌的特异性基因(invA、fliC、sdfⅠ)设计引物,优化反应体系和反应参数,建立多重PCR检测方法.应用该方法对国内17家鸡场369份病死鸡样品进行沙门氏菌的检测,检测结果与传统细菌培养法的结果进行比较.结果表明,优化过的多重 PCR 可同时对沙门氏菌种、属进行鉴定,灵敏度为4.6×102 CFU/mL;多重 PCR 方法共检测出沙门氏菌34株,其中鸡白痢沙门氏菌21株、肠炎沙门氏菌5株,与传统细菌培养法检测结果的符合率分别为91.2%、90.5%和80.0%.     

霍乱弧菌及副溶血弧菌复合PCR检测方法的建立

根据已发表的副溶血弧菌和霍乱弧菌的基因组序列,设计合成了三对引物,用于同时检测这两种弧菌。扩增片段长度分别为285 bp、450 bp、643 bp。经实验验证建立的PCR检测方法特异性较好,三对引物间无互相干扰;同时对建立的PCR检测体系进行了敏感性测定,结果最低检测下限为2.1×103cfu/mL。     

Analysis of rice blast resistance genes by QTL mapping

Resistance to rice blast pathogen mostly shows a quantitative trait controlled by several minor genes. Its complexity and the mutable characteristic of rice blast isolates both hinder the development of the blast resistance research. The article here tried to explore the resistance gene distribution on rice chromosomes and the way of function. Totally 124 QTLs have been identified against 20 isolates using Cartographer software with a ZYQ8/JX17 DH population, which separately are at 100 loci of 72 marker intervals on 12 rice chromosomes. Of them, 16 QTLs were determined by the isolate HB-97-36-1. 82 QTLs (66.13%) are from the resistant parent alleles, ZYQ8, while 42 QTLs (33.87%) are from the susceptible parent alleles, JX17. In comparison of their positions on chromosome, most QTLs are clustered together and distributed nearby the major genes especially the regions on chromosomes 1, 2, 8, 10 and 12. Each QTL could account for the resistance variation between 3~2%-68.64%. And, a positional QTL might display the resistance to several different isolates with different contributions.     

采用PCR技术检测植物病原菌

聚合酶联反应(Polymerase Chain Reaction ,PCR)用于各类DNA、RNA的体外扩增,具有灵敏、特异、快速等诸多优点,目前已广泛地作为人类、动植物病毒、真菌等分子检测的重要手段.并在此基础上与分子生物学其他最新技术相结合, 进一步发展了IC-PCR, real time fluorescent PCR, PCR-ELISA等复合PCR技术.将PCR技术与这些方法的突出优点相结合使用,从而提供了更快捷、特异、准确、高效检测侵染植物病原菌的方法.     

二重正交设计用于多重PCR条件优化

以猪圆环病毒Ⅰ,Ⅱ（PCVⅠ,PCVⅡ）为材料,探索多重PCR（multiplex PCR）最佳反应体系.结果表明,两次正交实验共34次反应,就获得了PCVⅠ,PCVⅡ多重PCR扩增的最佳条件组合,所得优化条件稳定,重复性好,说明正交法是一种快速,高效,经济的多重PCR条件优化手段。     

检测丽蚜小蜂取食寄主银叶粉虱的多重聚合酶链式反应

结合Ｄ２引物和ＢＥＦ引物,优化了一个可以用来检测丽蚜小峰（Ｅｎｃａｒｓｉａ ｆｏｒｍｏｓａ Ｇａｈａｎ）取食寄主银叶粉虱（Ｂｅｍｉｓｉａ ａｒｇｅｎｔｉｆｏｌｉｉ Ｂｅｌｌｏｗｓ＆Ｐｅｒｒｉｎｇ）的重多聚合酶链式反应。特异于银叶粉虱的ＢＥＦ引物（正向５′－ＡＡＴ ＴＣＡ ＣＡＣ ＡＴＡ ＣＧＴ ＴＡＧ ＣＣＣ ＣＴ－３′和反向５′－ＴＡＡ ＣＣＧ ＡＡＣ ＣＡＴ ＣＡＡ ＣＡＧ ＡＴＴ ＡＴＴ－３     

多重荧光PCR同时检测转基因成分35S和Nos方法的建立

根据商品化转基因作物中常用的花郴花花叶病毒启动子（CaMV35S）和根癌农杆菌终止（Nos）的序列特点，设计并合成了两对引物和相对应的荧光双链探针，建立一种应用荧光双链探针的多重荧光PCR同时检测转基因成分35S启动子和Nos终止子的方法，并利用该方法对马铃薯、大豆、玉米、甜椒、番茄等11份实物样品进行了检测，其中有5份样品结果阳性，结果表明所建立的多重荧光PCR方法能同时检测了35S方法同时检测出35S和Nos双组分，较常规PCR技术更为简便、快速、准确，有很很好的应用前景。     

中国鲎微卫星引物扩增圆尾鲎DNA的PCR反应体系优化

采用正交设计法,从dNTPs浓度、引物浓度、Mg2+浓度、Taq DNA聚合酶用量4个因素3个水平出发,优化设计圆尾鲎DNA的PCR反应体系(引物为中国鲎微卫星引物).并采用直观分析方法分析正交试验结果,最终建立了圆尾鲎SSR-PCR最佳反应体系:总体积20μL,Taq DNA聚合酶1.5 U、dNTPs 0.16 mmol/L、引物0.2μmol/L、Mg2+2.0 mmol/L;并通过PCR梯度实验进一步优化模板DNA质量浓度、退火温度及退火时间,获得最佳反应条件:模板DNA质量浓度为30 ng/μL,退火温度为48℃,退火时间为20～25 s.对最佳反应体系和反应条件进行了检验,结果显示该反应体系稳定性高、重复性好.     

Advances on methods for mapping QTL in plant

Advances on methods for mapping quantitative trait loci (QTL) are firstly summarized. Then, some new methods, including mapping multiple QTL, fine mapping of QTL, and mapping QTL for dynamic traits, are mainly described. Finally, some future prospects are proposed, including how to dig novel genes in the germplasm resource, map expression QTL (eQTL) by the use of all markers, phenotypes and micro-array data, identify QTL using genetic mating designs and detect viability loci. The purpose is to direct plant geneticists to choose a suitable method in the inheritance analysis of quantitative trait and in search of novel genes in germplasm resource so that more potential genetic information can be uncovered.     

Composite interval mapping of QTL for dynamic traits

The theoretical foundation for mapping quantitative trait loci (QTL) was laid by Sax[1] who discovered the association between the segregation pattern of pigment markers with seed size of bean. However, statistical methods were not well developed until th…     

Selection and development of representative simple sequence repeat primers and multiplex SSR sets for high throughput automated genotyping in maize

In the current study, 1900 maize simple sequence repeat (SSR) primers published in MaizeGDB were screened utilizing reference literature, 15 representative Chinese maize inbred lines and 15 Chinese maize hybrids from national regional testing. In total, 500 highly polymorphic primers were identified and used to construct a genetic map. 100 evenly distributed primers, 10 primers per chromosome, were further selected as a set of universal SSR core primers, recommended as preferred primers for general studies. These core primers were then redesigned and used to construct a high throughput multiplex PCR system based on a five-color fluorescence capillary detection system. We report here that two sets of ten-plex PCR combinations have been constructed, each consisting of 10 primers, with one primer per chromosome.     

鸡白痢沙门氏菌直接-多重PCR检测体系的建立

通过对鸡白痢沙门氏菌毒力质粒pSPUV上入侵质粒抗原J蛋白和质粒共转移调控因子的编码基因ipaJ和traJ的序列分析,在特异区域设计PCR引物,经沙门氏菌属不同种及常见肠道致病菌的交叉验证,筛选出三对鸡白痢沙门氏菌特异检测引物ipaJ-417、traJ-387、traJ-476.并且调试出一种利用两种DNA聚合酶的直接三重PCR检测体系:invA-211/traJ-387/16S-495,可以对鸡白痢沙门氏菌进行快速准确的鉴定.