大肠杆菌β-内酰胺酶耐药基因blaTEM,blaSHV,blaCTX-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,TEM blasHV和blaCTX-M)作为目的基因,设计3对特异性引物,建立了blaTEM,bla SHV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2.5 μL,MgCl2(25 mmol/L) 2.5 μL,dNTP (2.5 mmol/L)4 μL,blaTEM,blasHV和blaCTX-M上下游引物(25 μmol/L)分别为0.25 μL、0.5 μL、1.0μL,Taq DNA聚合酶(2.5 U/μL)0.4μL,模板2.5μL,反应总体积为25 μL.反应参数为:94℃5 min,94℃50 s,55℃55 s,72℃1 min,32个循环,72℃延伸5 min.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.     

春秋兼用家蚕品种“渝蚕1号”的育成

采用杂交育种和系统选择的方法,育成优良家蚕新品种中4、C106、日5,并与引进品种皓月组配成中4.C106×皓月.日5(渝蚕1号)的四元杂交组合。经四川省蚕品种审定的实验室鉴定和农村鉴定,该品种表现出强健、好养、发育整齐、茧丝质优良等特点,全茧量2.45 g、茧层率23.27%、解舒率75.22%,主要经济指标超过对照种。该品种已通过四川省家蚕品种审定,并在生产上试繁推广。     

日粮能量水平对肥育猪肌内脂肪含量、肌内和皮下脂肪组织脂肪酸组成的影响

以添加豆油方式提高日粮能量水平,研究其对肥育猪肌内脂肪(IMF)含量、肌内和皮下脂肪的脂肪酸组成的影响.36头杜长大母猪(平均体质量约40 kg)随机平均分为3组,分别饲喂3种能量水平(DE=12.82、14.24、15.66 MJ·kg-1,主要以添加豆油来提高日粮能量水平)的日粮.达到屠宰体质量(约90 kg)时每组屠宰5头,采取背最长肌和背部皮下脂肪,测定背最长肌IMF含量、肌内和皮下脂肪组织的脂肪酸组成.结果表明:不同能量水平日粮对IMF含量影响不显著(P>0.05);肌内和皮下脂肪的大部分饱和脂肪酸(SFA)和单不饱和脂肪酸(MUFA)含量随着日粮能量水平的提高而显著下降(P<0.05),大部分多不饱和脂肪酸(PUFA)含量随着日粮能量水平的提高而显著增加(P<0.05);IMF中PUFA(包括C18:3n6、C20:0、C20.4n6、C22:5n3)和C24:1的含量与IMF含量呈显著负相关;肌内和皮下脂肪组织中的大部分脂肪酸呈显著正相关,而少数脂肪酸(包括C18:3n6、CLA-c9t11、C20:0、C20:4n6、C22:5n3和C24:1)在2个组织中没有显著相关性;总体上看,2组织中的SFA、MUFA之间呈显著正相关,与PUFA呈显著负相关,PUFA之间呈显著正相关.本研究表明以添加豆油的方式提高日粮能苘量水平可以在不降低IMF含量的前提下改善猪肉的脂肪酸组成,从而提高猪肉的营养价值而不降低肉质.     

Sequence and Phylogenetic Analysis of rDNA ITS of Three Eimeria Species in Rabbit

In order to study whether the internal transcribed spacers (ITS) sequence could be used as a molecular marker for the species identification of rabbit coccidian, the rDNA ITS of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were amplified by polymerase chain reaction (PCR), and were cloned into pGEM-T Easy vector subsequently. The positive recombinant plasmids were identified by PCR and then sequenced. By sequence comparison and comparative analysis with the relative sequences of rabbit Eimeria spp. available in GenBank, the results showed that the lengths of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were 1065, 1009 and 1047 bp, respectively, and the sequence homologies with the same species sequences were 99.2%, 99.0% and 94.5%, respectively, while were 55.3% to 82.1% compared with corresponding sequences of other different species sequences. The phylogenetic analysis using software Mega 5.0 showed that all rabbit coccidia clustered together in a clade, which was divided into two sister lineages, corresponding to the presence or absence of oocyst residuum. The result demonstrated ITS could be used as a molecular marker for the species identification of rabbit coccidia.     

抗菌肽与抗生素的体外抗菌效果比较

应用由 Hancock实验室改进的微量肉汤稀释法测定了 cecropin P1、cecropin A、magainin 2、defensin 1、bactenecin、lactoferricin和 indolicidin等 7种抗菌肽和盐酸金霉素、去甲万古霉素、土霉素、强力霉素等 4种抗生素的体外抗菌活性 ,并且使用薄层琼脂糖孔穴扩散法比较了抗菌肽中 cecropin P1和 cecropin A与土霉素和呋喃唑酮对大肠杆菌 K88的抑菌效果。结果发现 ,抗菌肽与抗生素一样对几种革兰氏阳性菌和阴性菌都有不同程度的抗菌效果。其中 cecropin A、cecropin P1和 defensin1对大肠杆菌的 2个菌株 ATCC2 5 92 2和 K88的抗菌活性高于抗生素 ,defensin1是各种抗菌肽中对金黄色葡萄球菌抗菌效果最好的。抗生素对试验用金黄色葡萄球菌的抑制活性总体上要好于抗菌肽。与抗生素相比 ,cecropin A、cecropin P1和 indolicidin对猪霍乱沙门氏菌和鼠伤寒沙门氏菌具有更好的抗菌效果。另外 ,抗菌肽的抑菌圈边缘十分清晰 ,而抗生素的整个抑菌圈都比较模糊且界线不明。因此 ,从抗菌效果方面考虑 ,抗菌肽可以代替抗生素用于疾病的预防和治疗     

唇形科植物提取物对单胃动物的抗氧化作用及其机制

动物在患病、应激或特殊生理条件下,机体内会大量产生自由基,自由基与机体内的脂质、ＤＮＡ等产生反应,可对动物造成氧化损伤。唇形科植物提取物中的抗氧化物主要活性成分是酚类化合物,它和抗坏血酸及类胡萝卜素能够阻止自由基引发的氧化伤害。本文阐述了唇形科植物提取物对猪、鸡体外和体内抗氧化作用的实例与抗氧化功能,分析了其抗氧化作用机制。     

用卡片PCR法检测卡耶他环孢子虫

使用FTA卡片法提取卡耶他环孢子虫DNA,利用特异性引物组F1E/R2B、CC719/CRP999进行巢式PCR,得到1条298bp的特异条带。并以柔嫩艾美尔球虫、贝氏隐孢子虫等作为对照,结果显示,除卡耶他环孢子虫外,均无特异性条带。用卡耶他环孢子虫卵囊进行巢式PCR敏感性试验,结果显示敏感度最高达1.0×100个/mL。     

浓缩大豆磷脂对肉仔鸡生产性能的影响

本试验采用单因子随机化设计,将225只艾维菌肉仔鸡随机分为5个处理组,每个处理组设3个重复,每个重复15只鸡,以玉米-豆粕型日粮为基础日粮,饲喂处理1设为空白对照组;处理2添加猪油,设为猪油对照组;处理3、4、5分别添加低、中、高水平的浓缩大豆磷脂,为试验组.在0～3周龄猪油以2%,浓缩大豆磷脂分别以2%、3%、4%;在4～6周龄猪油以3%,浓缩大豆磷脂分别以3%、4%、5%添加到基础日粮中以替代相同比例的玉米,蛋白质水平维持不变.研究添加猪油和不同水平的浓缩大豆磷脂对肉仔鸡生长性能的影响,并对其作用机理进行了初步探讨.结果表明:在0～3周龄时,中、高水平试验组极显著地提高了采食量(P<0.01);在4～6周龄时,中水平试验组具有较大的采食量,但高水平试验组采食量有所下降;从全期来看,猪油对采食量影响不大;在0～3周龄,中、高水平试验组极显著地提高了增重(P<0.01),而且在此阶段添加油脂显著提高了饲料转化率(P<0.05);在4～6周龄时,添加油脂极显著提高了增重和饲料转化率(P<0.01);从全期来看,添加油脂可以提高增重与饲料转化率,且在0～3周龄时浓缩大豆磷脂优于猪油.     

Application of PCR-HRM in the study of relationship between leptin gene promoter polymorphisms and liver cirrhosis

AIM: To analyze the feasibility of PCR-high-resolution melting curve analysis (HRM) for detecting the site mutations of C2549 and G2548 in leptin gene promoter from the patients with liver cirrhosis, and to explore the relevance between mutant genotypes and physiological and biochemical indexes in liver cirrhosis patients. METHODS: Compared with the method of PCR-restriction fragment length polymorphism (RFLP), the present research used the method of PCR-HRM to analyze the site mutations of C2549 and G2548 in leptin gene promoter in control group (n=100) and liver cirrhosis group (n=100). The physiological and biochemical indexes of the patients were also detected and compared. RESULTS: Leptin gene promoter polymorphism was detected using PCR-HRM with effectiveness, high flux and accuracy. Preliminary results showed that the main mutation of the patients with liver cirrhosis was in C2549 site, but not found in G2548 site. Leptin, free leptin index (FLI), fasting insulin (FINS) and insulin resistance index estimated by homeostatic model assessment (HOMA-IR) in liver cirrhosis group were higher than those in control group. Insulin sensitivity index (ISI) and soluble leptin receptor (sOB-R) in liver cirrhosis group were lower than those in control group with significant difference except leptin level. Meanwhile, FLI showed positively correlated with FINS and HOMA-IR (r=0.45, r=0.53, P<0.05), and negatively with ISI (r=-0.34, P<0.05). In the patients with liver cirrhosis, C2549A heterozygous mutation was predominant. The indexes of HOMI-IR, leptin, sOB-R and FLI of C2549A homozygotes and heterozygotes were higher than those of the wildtypes, which showed significant difference except leptin and sOB-R levels (P<0.05). CONCLUSION: PCR-HRM can be more accurate for identifying leptin promoter polymorphism. The increase in the frequency of C2549A mutation may be closely related with liver cirrhosis. Existence of hyperinsulinemia and insulin resistance may be correlated with leptin level in the patients with liver cirrhosis.