参考群筛选方法及规模对基因型填充准确性的影响

为探究基于A矩阵期望遗传关系最大化（maximizing the expected genetic relationship for matrix A,RELA）、基于A矩阵目标群体遗传方差最小化（minimized the target population genetic variance for matrix A,MCA）、平均亲缘关系最大化（the highest mean kinship coefficients,KIN）、随机选择（random selection,RAN）、共同祖先筛选（common ancestor,CA）等不同参考群筛选方法及参考群规模对基因型填充准确性的影响。本研究使用矮小型黄羽肉鸡作为试验群体,采用鸡600K SNP芯片（Affymetrix Axion HD genotyping array）进行基因分型,测定435羽子代公鸡45、56、70、84、91日龄体重。利用Beagle软件将低密度SNP芯片填充为高密度SNP芯片数据,比较不同参考群筛选方法、参考群规模对基因型填充准确性的影响,以及填充芯片基因组预测准确性。结果表明,使用Beagle 4.0结合系谱信息进行填充效果最佳,其次为Beagle 4.0,而Beagle 5.1填充效果最差。使用MCA方法筛选参考群进行基因型填充准确性最高,使用RAN方法筛选参考群进行基因型填充准确性最低,MCA、RELA、CA 3种方法基因型填充准确性差别较小。相比其他方法,使用MCA方法筛选个体作为参考群将低密度SNP芯片填充至高密度SNP芯片进行基因组选择的预测准确性较高,与真实高密度SNP芯片的基因组预测准确性相差甚微。随着参考群规模增大,基因型填充准确性也随之增加,但增速逐渐下降,最后趋于平缓。综上所述,可以通过参考群筛选方法构建参考群以及控制参考群规模,以保证基因型填充和基因组预测准确性并节省成本,本研究为基因型填充在畜禽遗传育种中的应用提供技术参考。     

猪瘟野毒感染抗体阻断ELISA检测方法的建立与初步评价

以猪瘟野毒E2蛋白为包被抗原、辣根过氧化物酶标记的猪瘟野毒单抗作为酶标抗体,建立检测猪瘟野毒抗体的阻断ELISA方法。猪瘟野毒E2最适包被浓度为0.03μg/mL,待检血清最适稀释度为1∶4,酶标猪瘟野毒单抗稀释度为1∶1 000。用建立的阻断ELISA方法检测369份临床阴性血清,计算阻断率,确定临界值,阻断率>40%为猪瘟野毒抗体阳性,阻断率≤40%为猪瘟野毒抗体阴性。用建立的ELISA方法检测84份血清,其中78份为免疫猪瘟疫苗的血清,6份为猪瘟病毒感染血清。结果显示,78份免疫血清均检测为猪瘟野毒抗体阴性,6份猪瘟感染血清均检测为猪瘟野毒抗体阳性。因此可初步判定该方法可用于鉴别诊断猪瘟病毒自然感染动物和C株疫苗免疫动物的血清抗体,并为临床检测猪瘟野毒抗体提供便捷、快速,精准的检测工具,对猪瘟的临床诊断、预防以及猪瘟净化工作具有非常重要的参考意义。     

Molecular characterization of Ascaridia galli from Bangladesh and development of a PCR method for distinguishing A. galli from Heterakis spp.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.     

Recovery of sperms bearing X chromosomes with different concentrations of magnetic nanoparticles in ram

Pre-conceptual sex selection is still a highly debatable process whereby X and Y chromosome bearing spermatozoa are isolated before oocyte fertilization. Recently, magnetic nanoparticles (MNP) have been used to determine X and Y chromosomes bearing spermatozoa as a result of searching for a cheap, highly efficient method using non-toxic materials. This study aimed to recover the sperm bearing X chromosomes in ram with different concentrations of MNP and then evaluate the success of this method using polymerase chain reaction (PCR). Ram sperms were divided into four groups, treated with 0 (control), 50, 100 and 200 μg/ml MNP, respectively. MNP was used to restore sperm cells bearing X chromosomes. Upon recovery, the PCR was performed to identify the X and Y sperms, Methyl ThiazoleTetrazolium (MTT), to assess MNP toxicity and sperm viability and acridine orange (AO) to evaluate sperm DNA integrity. The results of PCR revealed that the treatment of spermatozoa- bearing X chromosomes with 50 μg/ml MNP had the highest effects on the recovery of X sperm rather than the other concentrations of MNP. However, the concentrations of MNP did not have any toxic effects on spermatozoa, sperm viability and, DNA integrity, but the high concentration of MNP (200 μg/ml) significantly reduced DNA integrity. According to MTT and AO results, the concentrations of MNP used in this study had no toxic effects on spermatozoa and did not reduce the sperm viability and DNA integrity, except that 200 μg/ml MNP significantly reduced DNA integrity.     

厄贝沙坦及其复方制剂中痕量杂质N-亚硝基-N-甲基-4-氨基丁酸残留方法学研究

采用超高效液相色谱-三重四级杆质谱(UPLC-MS/MS)建立厄贝沙坦及其复方制剂中N-亚硝基-N-甲基-4-氨基丁酸(NMBA)的测定方法。采用Waters ACQUITY UPLC○RHSS T3色谱柱进行分离,以0.005 mol/L甲酸铵(用甲酸调pH值至3.0)为流动相A,甲醇为流动相B,梯度洗脱,质谱检测器,流速为0.3 mL/min。结果显示NMBA的线性范围为0.156 6~3.132 6 ng/mL,相关系数r>0.99,方法检出限浓度为0.047 0 ng/mL(相当于样品浓度0.01μg/g),定量限浓度为0.156 6 ng/mL(相当于样品浓度0.03μg/g),样品加标回收率在80%~130%。说明该方法可以快速、简便、灵敏、准确地测定厄贝沙坦及其复方制剂中NMBA含量。     

生物酶法提取中草药活性成分研究进展

对酶提方法的原理、酶类选择依据、酶提工艺,以及酶提法应用现状进行了综述,旨在为同类研究提供技术参考。中草药具有广泛的应用前景和开发潜力,为提高中药疗效,方便临床用药,生产中常需要对中草药进行提取,然而传统的提取方法较为繁杂。随着科技进步,一些现代中药提取方法不断涌现。酶提方法是一种应用酶工程技术提取中药的现代中药提取方法,该方法具有高效、无毒、反应条件温和等优点,是一种发展潜力较大的中药提取方法。     

兽药中非法添加物检测及风险防范技术研究

本文总结了近年来兽药中非法添加的规律,分析成因;全面梳理现有检测标准,对非法添加物检测方法的检测对象和目标药物进行统计分析,研究检测方法发展及内在联系。在非法添加物检测方法提升和风险防范方面进行思考,提出筛查与确证方法并重、进一步扩大检测方法适用性、加强高效、高通量检测技术研究、拓展非法添加物检测品种范围;加强检查与宣传、疏堵结合,全面减少非法添加风险。     

发酵饲料中抑菌菌种筛选及抑菌性能评估方法的初步研究

试验通过牛津杯法筛选出抑制金黄色葡萄球菌和大肠杆菌性能较强的一株乳酸菌L3和两株芽孢杆菌D9、K9。将其用于制作发酵饲料,提升发酵饲料的抑菌性能。试验初步研究发酵饲料的体外抑菌性能评估方法。结果表明,用80%的乙醇溶液作为发酵饲料的前提取溶剂可增加抑菌物质的溶解,浓缩提取液的浓度可使发酵样品的抑菌效果更加明显。     

酸与碱处理对海带多糖提取及其抗氧化活性的影响

本试验采用柠檬酸提取法和碱提取法从海带中提取多糖组分,以总抗氧化能力和羟自由基（·OH）为衡量抗氧化活性指标,比较这两种提取方法得到海带粗多糖产率和抗氧化活性。试验结果表明：柠檬酸提取法和碱提取法的提取率分别为15.92%和6.05%,所提取的海带粗多糖具有良好的抗氧化活性,且柠檬酸提取法抗氧化活性更高。 [关键词] 柠檬酸提取法|碱提取法|海带多糖|抗氧化活性     

发酵乳中沙门氏菌依赖解旋酶恒温基因扩增快速检测方法的建立

建立依赖解旋酶恒温基因扩增（helicase-dependent isothermal DNA amplification,HDA）快速检测发酵乳中沙门氏菌方法。以沙门氏菌invA基因序列为目的基因,设计特异性引物,优化反应体系中UvrD解旋酶及T4 gp32添加量,建立最优反应体系。通过HDA方法直接检测发酵乳中沙门氏菌,扩增其产物后进行电泳检测,验证方法特异性。结果表明：采用HDA快速检测法检测发酵乳中沙门氏菌特异性良好,优化后反应体系体积50 μL时,UvrD解旋酶添加量为0.10 μg,T4 gp32添加量为5.0 μg,得到与设计序列长度（304 bp）一致的扩增产物,检出限为2.6×102 CFU/g;该方法用于快速检测发酵乳中沙门氏菌能够满足检测需求,具有较高的灵敏度、易操作,可作为一种基础且快速的方法检测发酵乳中沙门氏菌。