Establishment and Application of a Multiplex PCR System for the Detection of Blast Resistance Genes Pi-ta and Pi-b in Rice

Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas of China, which have been widely utilized in rice breeding and commercial production. In this study, on the basis of detection and verification of the genotypes of 22 rice varieties har- boring known blast resistance genes （Pi-ta and Pi-b） and blast susceptibility genes （pi-ta and pi-b）, two multiple PCR systems for these genes were established by us- ing the functional markers of blast resistance genes Pi-ta and Pi-b as well as blast susceptibility genes pi-ta and pi-b, respectively. Specifically, multiple PCR system I could simultaneously detect blast resistance genes Pi-ta and Pi-b, while system II could detect simultaneously blast susceptibility genes pi-ta and pi-b. In addition, the genotypes of 336 high generation breeding materials were detected with these two multiple PCR systems. The results were highly consistent with those of conventional single mark detection, indicating that these two multiplex PCR systems were stable, reliable and time-saving. The established multiplex PCR systems may serve as a rapid and efficient method to identify and screen rice germplasm resources and can be applied in marker-assisted selection to polymerize multiple genes for blast resis- tance in rice breeding.

A Single-Tube, Functional Marker-Based Multiplex PCR Assay for Simultaneous Detection of Major Bacterial Blight Resistance Genes Xa21, xa13 and xa5 in Rice

In marker-assisted breeding for bacterial blight(BB) resistance in rice, three major resistance genes, viz., Xa21, xa13 and xa5, are routinely deployed either singly or in combinations. As efficient and functional markers are yet to be developed for xa13 and xa5, we have developed simple PCR-based functional markers for both the genes. For xa13, we designed a functional PCR-based marker, xa13-prom targeting the In Del polymorphism in the promoter of candidate gene Os8N3 located on chromosome 8 of rice. With respect to xa5, a multiplex-PCR based functional marker system, named xa5 FM, consisting of two sets of primer pairs targeting the 2-bp functional nucleotide polymorphism in the exon II of the gene TFIIA5(candidate for xa5), has been developed. Both xa13-prom and xa5 FM can differentiate the resistant and susceptible alleles for xa13 and xa5, respectively, in a co-dominant fashion. Using these two functional markers along with the already reported functional PCR-based marker for Xa21(p TA248), we designed a single-tube multiplex PCR based assay for simultaneous detection of all the three major resistance genes and demonstrated the utility of the multiplex marker system in a segregating population.

Multiplex PCR Detection of Alleles Responsible for Benzimidazole- Susceptibility or -Resistance in Natural Populations of Haemonchus contortus

A multiplex PCR was developed to detect benzimidazole-resistance (BZ-R) or -susceptibility (BZ-S) in Haemonchus contortus by amplification with 4 primers of a sequence of the GRU-1 gene of β-tubulin of H. contortus making use of sequence information available in Genbank. The method was based on two allele-non-specific primers and two allele- specific primers. F1 (264 bp) and F3 (799 bp) should be produced in BZ-R, F2 (585 bp) and F3 in BZ-S. With this method, we demonstrated that H. contortus BZ-R strain from Australia showed F1 and F3, and the worm BZ-S strain from Shanghai did F2 and F3. Sequence analysis of the isotype 1 gene of β-tubulin of BZ-R from Australia and BZ-S from Shanghai showed the code in residue 200 of the gene was respectively TAC and TTC. The LD50 of albendazole of the Australian BZ- R strain was 0.54 μg mL-1, the Shanghai BZ-S strain was only 0.0023 μg mL-1 by EHA (egg hatch assay). The multiplex PCR could determinate the genotype of single adult worm or several third stage larvae and was performed on at least 50 ng of genomic DNA. BZ-R H. contortus were not detected in Shihezi and Yining of the Xinjiang, Wuhe of the Anhui Province, Nanjing and Xuzhou of the Jiangsu Province. The LD50 of the H. contortus from these locations to albendazole as determined by EHA varied between 0.0023-0.0032 μg mL-1. The result indicated that the multiplex PCR could be used to differentiate BZ-R and BZ-S of H. contortus and that the BZ-R situation of H. contortus was not serious in China.

Detection of the Mex Efflux Pumps in <i>Pseudomonas</i><i>aeruginosa</i>by Using a Combined Resistance-Phenotypic Markers and Multiplex RT-PCR

The aim of this study was to detect the expression of 4 clinically-important efflux pumps in the Resistance-Nodulation-Cell Division (RND) family including MexAB-OprM, MexXY, MexCD-OprJ and MexEF-OprN in Pseudomonas aeruginosa using a combination of resistance-phenotypic markers and multiplex RT-PCR (mRT-PCR). The antibiotic substrates specific for each Mex systems were used as phenotypic markers including carbenicillin, MexAB-OprM, erythromycin, MexCD-OprJ, norfloxacin and imipenem, MexEF-OprN and gentamicin, MexXY-OprM. The methods were validated with reference strains with known genotypes of the Mex systems and the potential applicability in clinical practice was tested with clinical isolates. The results for the reference strains support that the combination of resistance phenotype and mRT-PCR is a potential-attractive method for diagnosis of efflux-mediated resistance in P. aeruginosa. Further development to make it more practical for clinical use and study in a larger number of clinical isolates is required.

番茄抗根结线虫基因(Mi)和抗叶霉病基因(Cf5)的Multiplex-CAPS检测

建立了同时检测番茄抗根结线虫基因(Mi)和抗叶霉病基因(Cf5)的Mutiplex-CAPS技术,并利用该技术检测了12份番茄材料含有的抗性基因。

Tomato mottle mosaic virus: Characterization, resistance gene effectiveness, and quintuplex RT-PCR detection system

Tomato mottle mosaic virus(ToMMV), an economically important species of the genus Tobamovirus, causes significant loss in yield and quality of tomato fruits. Here, we identified the Shandong isolate of ToMMV(ToMMV-SD) collected from symptomatic tomato fruits in Weifang, Shandong Province of China. ToMMV-SD caused symptoms such as severe mosaic, mottling, and necrosis of tomato leaves, yellow spot and necrotic lesions on tomato fruits. The obtained full genome of ToMMV-SD was 6 399 nucleotides(accession number MW373515) and had the highest identity of 99.5% with that of isolate SC13-051 from the United States of America at the genomic level. The infectious clone of ToMMV-SD was constructed and induced clear mosaic and necrotic symptoms onto Nicotiana benthamiana leaves. Several commercial tomato cultivars, harboring Tm-2~2 resistance gene, and pepper cultivars, containing L resistance gene, were susceptible to ToMMV-SD. Plants of Solanum melongena(eggplant) and Brassica pekinensis(napa cabbage) showed mottling symptoms, while N. tabacum cv. Zhongyan 100 displayed latent infection. ToMMV-SD did not infect plants of N. tabacum cv. Xanthi NN, Brassica rapa ssp. chinensis(bok choy), Raphanus sativus(radish), Vigna unguiculata cv. Yuanzhong 28-2(cowpea), or Tm-2~2 transgenic N. benthamiana. A quintuplex RT-PCR system differentiated ToMMV from tomato mosaic virus, tomato brown rugose fruit virus, tobacco mosaic virus, and tomato spotted wilt virus, with the threshold amount of 0.02 pg. These results highlight the threat posed by ToMMV to tomato and pepper cultivation and offer an efficient detection system for the simultaneous detection of four tobamoviruses and tomato spotted wilt virus infecting tomato plants in the field.

桥臂复用型MMC的预充电控制策略

桥臂复用型模块化多电平换流器(AM-MMC)可以有效减少换流器的体积和重量,其预充电控制策略研究亟待开展。为此,提出了分别适用于交流和直流场景的AM-MMC的预充电控制策略。在交流预充电场景下,通过分析桥臂间子模块电容电压的关系,并考虑子模块电容过压以及桥臂间子模块电容电压不均衡问题,提出一种包含不控充电阶段、可控充电阶段和阻断阶段的三阶段交流预充电控制策略,并提供了限流电阻的计算方法。在直流预充电场景下,推导冲击电流产生的原因,提出了一种基于直流电流的冲击电流抑制策略。基于MATLAB/Simulink的仿真结果表明,所提预充电控制策略可有效实现AM-MMC的平稳启动。

A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance

CRISPR homing gene drives have considerable potential for managing populations of medically and agriculturally significant insects.They operate by Cas9 cleavage followed by homology-directed repair,copying the drive allele to the wild-type chromosome and thus increasing in frequency and spreading throughout a population.However,resistance alleles formed by end-joining repair pose a significant obstacle.To address this,we create a homing drive targeting the essential hairy gene in Drosophila melanogaster.Nonfunctional resistance alleles are recessive lethal,while drive carriers have a recoded“rescue”version of hairy.The drive inheritance rate is moderate,and multigenerational cage studies show drive spread to 96%–97%of the population.However,the drive does not reach 100%due to the formation of functional resistance alleles despite using four gRNAs.These alleles have a large deletion but likely utilize an alternate start codon.Thus,revised designs targeting more essential regions of a gene may be necessary to avoid such functional resistance.Replacement of the rescue element’s native 3'UTR with a homolog from another species increases drive inheritance by 13%–24%.This was possibly because of reduced homology between the rescue element and surrounding genomic DNA,which could also be an important design consideration for rescue gene drives.

98株鸭源致病性大肠杆菌氨基糖苷类耐药基因型与耐药表型的比较

旨在调查鸭致病性大肠杆菌氨基糖苷修饰酶耐药基因(AMEs基因)的携带情况,探讨耐药基因与氨基糖苷类抗生素耐药表型的相关性。对98株鸭致病性大肠杆菌采用了K-B法,选用氨基糖苷类抗生素链霉素、新霉素、庆大霉素、阿米卡星、大观霉素和卡那霉素进行药敏试验,用建立的检测氨基糖苷类AMEs主要基因的四重PCR方法对上述菌株进行分子检测,并随机选取耐药基因ant(3″)-Ⅰa、aac(3)-Ⅱa和aph(3′)-Ⅱa各3个阳性扩增进行克隆测序,对药敏试验结果和基因检测结果进行比较分析。结果表明,98株鸭致病性大肠杆菌有67株对上述氨基糖苷类药物中的一种或多种耐药,耐药率为68.4%(67/98);有49株扩增出AMEs基因,AMEs基因的检出率为50%(49/98),其中ant(3″)-Ⅰa的检出率为30.6%(30/98),aac(3)-Ⅱa为13.3%(13/98),aph(3′)-Ⅱa为3.1%(3/98),ant(3″)-Ⅰa+aac(3)-Ⅱa为2.0%(2/98)、aac(3)-Ⅱa+aph(3′)-Ⅱa为1.0%(1/98),未检出aac(6′)-Ⅰb基因;序列分析结果表明,扩增产物与GenBank中的相应序列有很高的同源性(>99%);AMEs耐药基因与耐药表型的符合率为73.1%(49/67),符合率从高到低依次为大观霉素60%(3/5)、庆大霉素55%(11/20)、链霉素33.3%(22/66)、卡那霉素19%(4/21)、新霉素12.5%(1/8)、阿米卡星0%(0/3)。另外有4株细菌检测到相关耐药基因但耐药表型为敏感,而有22株耐药表型为耐药却未检测到相关耐药基因。鸭致病性大肠杆菌氨基糖苷类耐药基因以ant(3″)-Ⅰa和aac(3)-Ⅱa两种为主,耐药性与相关耐药基因的检出率基本呈正相关。

多重聚合酶链反应检测结核菌耐异烟肼相关基因

目的 :建立耐异烟肼 (isoniazid ,INH)结核分枝杆菌(M .tuberculosis,MTB)多重聚合酶链反应 (multiplePCR ,multi PCR)检测系统 ,在一次扩增中快速 ,特异地同时检出aphC启动子 ,inhA和kagG基因 ,用于快速初步诊断结核分枝杆菌对INH的耐药性 .方法 :根据结核分枝杆菌的aphC启动子 ,inhA和kagG序列 ,分别设计出 3对特异性寡聚核苷酸引物 ,采用multi PCR技术 ,同时检出对结核分枝杆菌耐INH起作用的 3个基因 .结果 :应用multi PCR反应体系 ,对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果 ;multi PCR扩增的预期结果为 :单基因引物出现一条特异性扩增区带 ,多重基因引物出现 2或 3条特异性扩增区带 ,经实验达到预期的扩增结果 ;对H3 7Rv标准株、INH敏感株及INH耐药株分别采用常规PCR和multi PCR进行同时扩增 ,结果两种扩增方法均能扩增出预期的目的片段 ,符合率达 10 0 % .结论 :multi PCR能有效地为多基因耐药的临床病原体的诊断提供快速、准确的诊断手段 。

转基因抗草甘膦油菜的实用PCR检测方法

利用定性PCR技术 ,建立了检测孟山都公司培育的转基因抗草甘膦油菜中的epsps、gox和fmv35s的方法 ,同时设立了阳性内对照napin。该方法的检测灵敏度为 0 .0 5 %。利用复合PCR可以在一个PCR反应中同时检测出epsps、gox、fmv35s和napin基因。这种方法可以有效地用于转基因抗草甘膦油菜中的外源基因的检测。

江苏奶牛空肠弯曲菌和结肠弯曲菌流行状况及耐药性分析

目的了解江苏省奶牛空肠、结肠弯曲菌流行及耐药状况。方法采用多重PCR方法对10个奶牛场的产奶牛、育成牛和饲养环境进行空肠弯曲菌和结肠弯曲菌流行状况调查,采用琼脂扩散法测定分离株的耐药性。结果1531份样品中,119份空肠弯曲菌阳性,平均阳性率7.77%;3份结肠弯曲菌阳性,平均阳性率0.20%;空肠弯曲菌、结肠弯曲菌阳性率最高分别为30.91%、3.57%。在三类样品中,产奶牛空肠弯曲菌、结肠弯曲菌阳性率分别为5.02%、0.32%,育成牛空肠弯曲菌阳性率为8.70%、未检出结肠弯曲菌,环境样品空肠弯曲菌、结肠弯曲菌阳性率为10.28%、0.18%。35株奶牛空肠弯曲菌分离株对8大类21种抗生素高度敏感率的是:阿莫西林100%、阿齐红霉100%、链霉素97.14%、庆大霉素94.29%、红霉素91.43%、头孢噻肟82.86%、克林霉素82.86%;高度耐药率的是:头孢哌酮100%、恩诺沙星100%、环丙沙星97.14%、复方新诺明97.14%、左旋氧氟沙星94.29%、萘啶酸94.29%、头孢拉定94.29%、诺氟沙星91.43%、头孢克罗88.57%。菌株耐药谱显示,35株分离株的耐药主要集中在9耐到12耐,占88.57%,产奶牛分离株的多重耐药性较其它分离株更严重。结论我国奶牛群中空肠弯曲菌、结肠弯曲菌的流行和耐药状况呈现多样化和复杂化,研究结果为正确评价我国奶牛群弯曲菌的流行状况和制定切实可行的防控措施提供科学依据。

水稻抗稻瘟病基因Pi-ta和Pi-b多重PCR体系的构建与应用

稻瘟病是我国水稻主产区的重要病害之一,其主效抗性基因Pi-ta和Pi-b在我国很多稻区表现广谱持久的稻瘟病抗性,被广泛应用于我国的水稻育种和生产。本研究选用稻瘟病抗性基因Pi-ta和Pi-b及其等位基因的功能标记,在对22份分别已知抗病基因Pi-ta和Pi-b以及感病基因pi-ta与pi-b组成的水稻品种检测验证基础上,建立了2套稻瘟病基因多重PCR体系:体系I同时检测抗病基因Pi-ta与Pi-b,体系II同时检测感病基因pi-ta与pi-b,并利用2套体系对336份高世代育种材料进行检测,与单标记检测结果比较,表现稳定可靠,重复性好。本研究构建的抗稻瘟病基因分子标记多重PCR体系可用于水稻种质资源的快速评价和抗稻瘟病分子标记辅助育种。

多重PCR检测食源金黄色葡萄球菌nuc、blaZ和mecA基因方法的建立与应用

目的建立快速检测食源金黄色葡萄球菌(Sa)耐热核酸酶基因(nuc)、β-内酰胺酶基因(blaZ)和甲氧西林耐药基因(mecA)的多重PCR方法。方法根据GenBankSa的nuc、blaZ和mecA基因序列,设计3对特异性引物,建立鉴定Sa及分析Sa耐β-内酰胺类抗生素三重PCR方法 ,并对79株食源Sa进行基因检测与表型比较。结果显示供试Sa中检出nuc、blaZ基因阳性率分别为97.47%、73.42%,mecA基因检出为阴性;nuc基因检出与其表型符合率达97.47%;而blaZ扩增结果与β-内酰胺酶试验、青霉素类敏感试验同时符合率为49.37%,前者与后两者符合率分别为60.76%和75.95%;mecA与耐甲氧西林表型符合率为100%;此次79株食源Sa中无耐甲氧西林Sa。结论该方法简便、快捷、准确,为食源Sa鉴定和耐药性分子分析提供了参考依据。

大肠杆菌氯霉素类耐药基因三重PCR检测试剂盒的研究与应用

本研究研制了大肠杆菌氯霉素类药物耐药基因cat1、flor、cmlA基因三重PCR检测试剂盒。结果显示,该试剂盒具有良好的重复性、灵敏度以及保存期长等特点。应用该试剂盒检测107株猪源鸡源大肠杆菌,cat1、flor、cmlA三种基因的检出率分别是41.1%、51.4%和36.4%。本试剂盒对氯霉素类药物耐药基因的传播、流行调查以及在临床用药方面具有重要的指导意义。

大肠杆菌β-内酰胺酶耐药基因bla_(TEM),bla_(SHV),bla_(CTX-M)三重PCR检测方法建立

本研究以我国大肠杆菌中常见的3种β-内酰胺酶耐药基因(bla,TEMblaSHV和blaCTX-M)作为目的基因,设计3对特异性引物,建立了blaTEM,blaSHV和blaCTX-M耐药基因三重PCR检测方法。三重PCR反应体系为:10×PCR缓冲液2.5μL,MgCl2(25mmol/L)2.5μL,dNTP(2.5mmol/L)4μL,blaTEM,blaSHV和blaCTX-M上下游引物(25μmol/L)分别为0.25μL、0.5μL、1.0μL,Taq DNA聚合酶(2.5U/μL)0.4μL,模板2.5μL,反应总体积为25μL。反应参数为:94℃5min,94℃50s,55℃55s,72℃1min,32个循环,72℃延伸5min。本方法的建立为大肠杆菌中β-内酰胺酶耐药基因的快速检测及分子流行病学调查提供了新的手段。

沙门菌耐药基因多重PCR检测方法的建立

目的建立检测沙门菌耐药基因的多重PCR方法。方法在已有的单一PCR检测沙门菌耐药基因的基础上,采用正交试验设计法,对Mg2+、dNTPs和引物浓度比例以及退火温度进行优化,确定沙门菌耐药基因多重PCR最佳反应体系和条件,并运用多重和单一PCR同步比较检测细菌耐药基因。结果成功建立了氨基糖苷类药物耐药基因aph(3)-IIa、aadA2、aacC4三重PCR方法、氯霉素类药物耐药基因flor、cmlA和catI三重PCR方法、四环素类药物耐药基因tetA和tetB二重PCR方法,且多重和单一PCR同步检测相关耐药基因结果符合率达100%。结论建立的多重PCR具有特异、快速、简便的特点,可为沙门菌耐药基因的检测以及耐药性的监控提供技术支撑。

小麦黄矮病品种抗病性调查及种群多重PCR检测

为了解当前大面积种植小麦品种及种质资源对小麦黄矮病(BYDV)的抗病性,在利用自然发病和人工接种的方法对小麦抗病性进行鉴定的基础上,构建了可同时检测病原BYDV三个不同种群的多重PCR体系,并检测了陕西省杨凌地区的种群发生状况以验证体系的稳定性。结果表明,2007年田间BYDV自然发病率高达86.5%,2008年和2009年发病率降低,分别为10.6%和14.0%,不同年份间自然发病率差异大。2009年在自然发病的基础上进行人工接种鉴定,发现品种和资源发病率较自然发病率升高43.7个百分点,而已感病材料的发病率变化不大。小冰麦×小偃22(F_1)、小偃22×小偃6号(F_1)、石5144×小偃22(F_2)等分离群体和小冰麦、豫麦34等小麦品种部分植株感病初期发病级别分别达到3、2、5、5、6级,而后期级别分别降至0、0、1、2、0级,表现出恢复现象。小偃6号、小偃54、小偃597等小偃系列品种发病率高,2009年人工接种后小偃系列品种发病率高达总调查材料数(157种)的13.5%。秦丰148、西农402、陕715、西农979四个品种连续三年表现抗病。利用多重PCR体系检测了杨凌地区的8个田间自然发病样品,发现混合感染现象严重,PAV感染率达到25%,也证明多重PCR体系稳定可靠。

鸭源大肠杆菌3种tet基因的多重PCR检测

利用肉汤稀释法测定21株鸭大肠杆菌对四环素等7种抗菌药物的敏感性,并用多重PCR方法同时检测3种四环素耐药基因(tetA、tetB和tetC)。结果显示:所有菌株均呈现tetA、tetB和tetC基因阳性,与药敏试验结果阳性符合率76.67%,携带3种tet基因的临床分离鸭大肠杆菌不仅对四环素类药物耐药,还对头孢噻肟、恩诺沙星和氟苯尼考耐药,呈现多重耐药的特点。

耐碳青霉烯鲍曼不动杆菌的耐药机制及分子流行病学

目的碳青霉烯耐药鲍曼不动杆菌(CRAB)逐年升高的分离率和全球播散已成为极为严重的问题,本研究目的是阐明我院CRAB的耐药机制和分子流行病学特征。方法使用gyrB多重PCR方法将我院2014年7月—2015年6月分离的327株非重复醋酸钙-鲍曼不动杆菌复合体(ABC)鉴定到种,VITEK-2仪器法和E-test法测定抗菌药物的最低抑菌浓度(minimal inhibitory concentration MIC),PCR方法检测常见的碳青霉烯酶耐药基因、ISAba1并测序,Turton 2组多重PCR方法进行分子分型。结果327株ABC中有315株鲍曼不动杆菌(ABA)、9株皮特不动杆菌和3株医院不动杆菌,ABA、皮特和医院不动杆菌对亚胺培南的耐药率分别为91.7%、0和66.7%,耐药株主要分离自急诊科和呼吸ICU。ABA bla_(OXA-23-like)和ISAba1检出率均为91.1%,测序均为bla_(OXA-23),bla_(OXA-51-like)和ISAba1检出率分别为100%和0.3%,测序主要为bla_(OXA-66)和bla_(OXA-69),未检测到其他OXAs型、NDM、IMP、GIM、KPC、SIM、VIM和GES酶基因。Turton分子分型76.8%属于国际克隆Ⅱ型(IC Ⅱ)。结论携带bla_(OXA-23)和ISAba1的IC Ⅱ克隆是我院主要的流行克隆株,克隆播散是CRAB感染增加和暴发的重要原因。