Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China

The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP) assay and to research the recent epidemiology of contagious ecthyma in Jilin Province, China, using the assay. A LAMP assay targeting a highly conserved region of the F1L gene was developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five cases from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were tested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was 100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR. No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. The average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged 1–6 months. Our results demonstrated that CEV infection was very widespread in the flocks of Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitive detection of CEV infection.     

甜菜DAMD-PCR体系的建立及优化

为了建立甜菜DAMD扩增体系,以期利用DAMD引物应用于甜菜品种指纹图谱的构建及分子标记辅助育种。本实验利用单因素变量的方法对甜菜DAMD体系进行优化。同时选用12个甜菜品种,利用优化的体系对25条DAMD引物进行扩增。获得甜菜的最适DAMD体系:总体积为20μL,包含模板DNA 10~80 ng、0.75 U的DNA聚合酶、0.2μL的d NTPs(2.5 mmol/L each)以及2.0μL的引物(10μmol/L)。同时25条引物均扩增出了清晰条带,除了个别引物多态性较差外,其余引物多态性都非常的丰富,其中引物62H(-)就可以把实验中用到的12个甜菜品种全部区分开。由此可见,DAMD引物的扩增效率很高,并且扩增结果稳定,条带清晰,非常适合甜菜品种指纹图谱的构建及遗传多样性分析。     

柴达木盆地梭梭LEA基因的扩增及序列分析

为发掘梭梭(Haloxylon ammodendron)抗逆相关基因,以柴达木盆地梭梭的同化枝为材料,扩增其LEA基因并进行了序列测定与分析。结果表明,所得柴达木盆地梭梭的LEA基因碱基数为234 bp,其中A碱基70个,T碱基52个,G碱基59个,C碱基53个;氨基酸序列分析表明,所得LEA基因片段编码78个氨基酸,其中强碱性氨基酸有12个,强酸性氨基酸有6个,疏水性氨基酸有34个,亲水性氨基酸有26个。     

白菜型油菜DFR基因的克隆与序列分析

By using RACE (rapid amplification ofcDNA ends) based homologous cloning strategy, we have successfully isolated the genomic and full-length cDNA sequences of a gene encoding typical DFR (dihydroflavonol-4-reductase) from black-seeded Brassica campestris L. var. oleifera DC.. The gene, designated BcDFR here, is 1 722bp in length and harbors 5 introns with typical splice sites of plant DFR genes. BcDFR cDNA is 1311bp in length with a 1 158bp ORF as well as a 25bp 5‘ UTR and a 128bp 3‘ UTR. The encoded BcDFR protein is 385 aa with a calculated Mw of 42.85kD and a pI value of 5.55. The nucleotide and amino acid sequences of this gene share extensive homologies to plant DFR genes of wide origins especially high similarities to Cruciferous DFR genes. Sequence analyses such as phylogenetic analysis, conserved domain search and substrate specificity region detection all indicated that BcDFR gene is a quite potentially biofunctional gene. Its cloning enables us to further dissect the possible relatedness between DFR gene and Brassica seed coat color traits and to create transgenic novel yellow-seeded rapeseed germplasm through antisense- or RNAi-suppression of DFR gene expression in black-seeded elite cultivars.     

Isolation and characterization of microsatellites in Bambusa arundinacea and cross species amplification in other bamboos

Isolation and characterization of microsatellites was analysed in Bambusa arundinacea and cross species amplification studied in other bamboos. Microsatellites, tandem repeats of short nucleotide (1–6 bp) sequences, are the DNA marker of choice because of their highly polymorphic, ubiquitous distribution within the genome, ease of genotyping through Polymerase chain reaction, selectively neutral, co‐dominant and multiallelic nature. Six microsatellites, three polymorphic and three monomorphic have been characterized for the first time in a bamboo species, Bambusa arundinacea belonging to the family Poaceae. The numbers of alleles per locus ranged from 2 to 13. Cross species amplification was tested in 18 other bamboo species. Monomorphic simple sequence repeats (SSRs) were found to be cross amplified in most of the species tested and polymorphic ones in only three to four species. The utility of SSR loci in a genetic diversity study of B. arundinacea and other cross‐amplified bamboo species has been discussed. This study will help in population genetic studies in bamboo species.     

Evaluation of 'quick and dirty' DNA extraction methods for marker-assisted selection in rice (Oryza sativa L.)

Six simple methods for extracting DNA from rice seedlings were evaluated for marker‐assisted selection (MAS). The assessment of each method was based on PCR amplification of SSR markers, DNA yield and purity, time and cost. Based on these criteria, two methods were selected as being superior to other methods. The best two methods included the standard method developed at the International Rice Research Institute (IRRI), which utilizes a sodium dodecyl sulfate extraction buffer followed by chloroform/isoamyl alcohol extraction and a previously published method using sodium hydroxide and Tris. These two methods produced nearly identical PCR amplification results. The sodium hydroxide method is considerably simpler, quicker and cheaper than the standard IRRI method, and may be particularly useful for many applications of MAS or high‐resolution mapping. This method was also adapted into an effective high throughput method utilizing 96‐well plates emphasizing its versatility.     

Effect of Soil Organic Matter on Adsorption of Nitrification Inhibitor Nitrapyrin in Black Soil

ABSTRACT

Experiments were conducted to study the adsorption kinetics and isothermal adsorption mechanism of nitrification inhibitor nitrapyrin onto the black soil of northeastern China. The nitrapyrin adsorption kinetics of the black soil were fitted with a quasi-second-order kinetics equation before and after organic matter removal (R² ≥ 0.8320, p < .05). The adsorption isotherm was obtained by the Langmuir equation (R² ≥ 0.9400, p < .05). Isothermal adsorption requires less energy and is a spontaneous endothermic reaction with an L-type isothermal curve (n > 1), dominated by surface physical adsorption. With the decrease of organic matter content in black soil, the adsorption mechanism of nitrapyin changed from distribution mechanism to surface adsorption. The black soil’s organic matter content exhibited a quadratic relation with the maximum adsorption capacity and adsorption constant (Kf) at the different temperatures, reaching significant levels (R² ≥ 0.6484, p < .05).     

环介导恒温扩增技术在动物病原检测中的应用

环介导恒温扩增技术(LAMP)是一种新型核酸扩增技术,是依赖于靶目标上6个区域的4条引物和一种具有链置换活性的DNA聚合酶Bst的作用,在60 ℃～65 ℃恒温条件下1 h之内即可完成的基因扩增技术.该技术虽然起步较晚,但发展迅速,目前在很多领域的研究和应用已经取得很大进展.论文从技术原理、引物设计、反应机制和特点等方面了介绍了环介导恒温扩增技术,并就该技术在动物细菌性病原微生物、病毒和寄生虫检测中的应用做了介绍.     

猪繁殖与呼吸综合征病毒M基因RT-LAMP检测方法的建立

本研究根据GenBank中登录的猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)M基因保守序列6个特异性部位设计了 2对引物,建立了PRRSV的环介导逆转录等温检测方法。结果表明,该方法灵敏度比RT-PCR高100倍;全部反应可在1 h内完成并可得到其特有的阶梯状条带,而且猪圆环病毒、猪瘟病毒、猪伪狂犬病毒的扩增结果均为阴性;在反应体系中添加SYBR GREENⅠ染料后,可通过肉眼观察有无荧光而直接判定结果。该方法灵敏度高、特异性好,可作为猪繁殖与呼吸综合征病毒的快速诊断方法。     

不同动物部分组织基因组甲基化程度的差异分析

应用甲基敏感扩增多态性(MSAP)技术,检测和分析了猪、牛、羊、小鼠、鸡和鸭基因组的甲基化程度。结果表明,对CCGG位点,实验中检测的几种动物的甲基化程度多数在0.40￣0.50之间(不包括牛);不同动物来源相同组织基因组的甲基化程度不同,相同动物不同组织基因组的甲基化模式具有特异性;同一种动物,组织基因组的甲基化程度一般都高于血液基因组;另外,哺乳动物与禽类基因组甲基化程度未发现较大的差别,但哺乳动物基因组全甲基化位点较多,半甲基化位点较少。