用磁珠富集法从AFLP片段中分离鹅微卫星DNA标记

用磁珠富集法从AFLP片段中分离微卫星DNA标记，可以避免基因组文库的构建和筛选这一繁重的工作，既节省时间又节约费用。利用少量的内切酶组合建立一系列AFLP DNA，然后用生物素标记的简单重复序列（SSR）作探针与其杂交，杂交复合物固定到包被有链亲和素的磁珠上，经过一系列洗涤过程，含有SSR的AFLP片段被吸附到磁珠表面。这些片段经洗脱下来后，先用对应的AFLP引物扩增，再进行克隆和测序，根据SSR两端的保守序列设计引物，经过多态性分析后，便可得到微卫星DNA标记。用这种方法已成功地从6个中国地方品种鹅中分离到30个微卫星DNA标记。其中有11个引物表现为多态性好，特异性强且相互之间并不连锁。各品种平均多态信息含量（PIC）在0．513～0．546之间。说明这11个微卫星均为高度多态，可作为有效的遗传标记用于鹅品种间遗传多样性和系统发生关系的分析，并为建立鹅遗传图谱、重要经济性状的基因定位及标记辅助选择奠定基础。     

从江香猪基因组甲基化修饰与体重之间的相关性研究

为了探索从江香猪生长慢的表观遗传学机理,本试验应用甲基化敏感扩增多态性(methylation-sensitive amplifying polymorphism,MSAP)技术,将60头20 d从江香猪分为高体重组和低体重组,研究血液和肝脏基因组DNA中胞嘧啶的甲基化程度,并测定差异的甲基化片段核苷酸序列.结果发现,MSAP分析得到5 977条带;与低体重组相比,高体重组的血液和肝脏总甲基化水平较低,两组的血液半甲基化水平均较肝脏中的高,全/总甲基化水平较低.提示香猪血液和肝脏基因组的总甲基化程度随体重的增加而下降.经克隆测序得到5条有差异的甲基化条带A1-A5,其中A1和A2源自血液,A3-A5来自肝脏;A1和A4片段为高体重组特有,其余3条片段为低体重组特有;A3片段未找到相关基因,其他4个片段处于代谢或免疫相关的基因内部或5'侧翼区.提示从江香猪基因组的甲基化水平存在个体差异,并与20 d体重相关;得到的4条甲基化片段,将有助于香猪甲基化调控机制的研究.     

Genetic evaluation of IS900 partial sequence of Mycobacterium avium subsp. paratuberculosis Brazilian isolates from bovine milk

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis. Insertion sequence IS900 is used for the identification of MAP. The objective of this study was to verify the genetic conservation of IS900 sequences in raw milk samples. To evaluate genetic conservation, 206 quarter milk samples and 16 bulk-tank milk samples were collected. DNA extraction and IS900 PCR were performed in all samples. Six samples amplified the expected fragment. To confirm the identity of the amplified fragments, PCR products were cloned and sequenced. The resulting sequences were compared with other MAP sequences from GenBank, and it was possible to identify eight polymorphic regions and to form five distinct haplotypes. The number of mutations in each haplotype was verified. IS900 sequence is a very well-conserved sequence that could be used as tool for the molecular detection of this agent and epidemiological purposes. The results showed the first genetic analysis on Brazilian isolates of MAP.     

基于线粒体12S rRNA基因序列鉴别牛肉的种源

 在提取黄牛肉、牦牛肉和水牛肉总DNA的基础上,设计通用引物进行PCR扩增,电泳回收PCR产物后双向测序,再通过构建系统进化树鉴别牛肉的物种来源。PCR扩增获得的牦牛、水牛12S rDNA基因片段大小都为440 bp,黄牛12S rDNA基因片段大小都为439 bp。参照引用的不同牛种12S rDNA基因序列,构建的系统进化树能够清晰地鉴别测序样品的牛种来源。因此,结合运用PCR扩增和DNA测序技术是一种精确可靠的方法,能够有效地运用于牛肉的种源鉴别。     

基于粪便样本的野生犬科动物物种鉴别与棘球绦虫感染调查

旨在探索粪便样本分析对犬科动物物种鉴别的方法及其可行性,同时对野外犬科动物棘球绦虫感染情况进行调查。采用哺乳动物线粒体DNA控制区通用引物对101份采集自野外环境的犬科动物粪便DNA进行PCR扩增和测序,通过核苷酸位点变异分析、BLAST相似性比对、遗传距离分析和系统进化树构建,对粪样来源物种进行分子鉴别。同时,采用粪-抗原ELISA检测,对粪样棘球绦虫感染情况进行调查。结果表明：粪样来源物种鉴别的检测成功率为73.27%,获得的74条序列共定义单倍型20个,所有单倍型序列均可在GenBank数据库中匹配到单一物种,序列相似度为98.52%~100%。单倍型根据序列差异可分为4个单倍型集,各单倍型集间平均碱基差异为17.83~70.10,各单倍型集内单倍型碱基差异在1~10个之间。各集合间遗传距离为0.068~0.342,远大于各集合内单倍型间遗传距离0.009~0.022。系统进化分析结果显示,同一集合的单倍型以高自展值聚集成一支。综合各项分析结果,可推测所检测粪样宿主来源分别为犬、灰狼、赤狐和红狐。粪-抗原ELISA检测发现阳性样本11份,样本棘球绦虫抗原阳性率为10.89%。综上表明：线粒体DNA控制区序列分析可用于犬科动物的分子鉴别,可结合核基因等其他分子标记进一步提升检测准确率。研究区域野外犬科动物棘球绦虫粪抗原阳性率较高。     

Molecular characterization of the ITS-2 fragment of Paramphistomum leydeni (Trematoda: Paramphistomidae)

Paramphistomosis has shown an increased spread over the last years in Argentina, being in some regions an emerging parasitosis, which has motivated their study and identification. Even when morphological features were reported, molecular characterization appears as a complementary and reliable tool. Samples of Paramphistomum leydeni collected since 2002 from different origin, definitive hosts, natural or experimental infections and preservation method were subjected to PCR-RFLP for ITS2+ fragment, and compared to Fasciola hepatica and Notocotilidae cercarie. Frozen and ethanol fixed samples amplified fragments of 500 bp for P. leydeni and Notocotilidae samples, while F. hepatica eggs produced a 540 bp amplicon. Restriction fragments obtained from endonucleases HinfI, HhaI, BsuRI, TaqI, and TruI were identical for all P. leydeni samples, supporting the morphological classification previously performed. Four selected amplicons were sequenced and reported at GenBank, given a consensus sequence ITS-2+ of 441 bp. This first report of molecular characterization for P. leydeni, improves the current knowledge of the genus and establishes precedents for further specimen classifications.     

Evaluation of four DNA extraction methods for the detection of Tritrichomonas foetus in feline stool specimens by polymerase chain reaction

Feces are increasingly valued as practical samples for molecular diagnosis of infectious disease. However, extraction of polymerase chain reaction (PCR) quality DNA from fecal samples can be challenging because of coextraction of PCR inhibitors. Because the type and quantity of PCR inhibitors is influenced by diet, endogenous flora, and concurrent disease, it is unlikely that extraction method performance with human feces can be directly extrapolated to that of domestic cats. In the present study, 4 commercially available DNA extraction methods were examined for their influence on the sensitivity of PCR for the detection of Tritrichomonas foetus in feline stool. DNA was extracted from serially diluted feline-origin T. foetus trophozoites in the absence or presence of feline feces. The ZR Fecal DNA kit was identified as affording the greatest analytical sensitivity and reproducibility and was able to detect >/=10 T. foetus organisms per 100 mg feces in 100% of PCR reactions. Further, the identified extraction method could be completed in the shortest time of all kits tested.     

基于ISSR分子标记分析陆川猪种群遗传差异

试验旨在探究陆川猪种群之间的遗传差异,对陆川猪种群进行遗传多样性分析,为种质改良与创新提供参考。以29份陆川猪种及部分广西地方猪种样品为试验材料,采用了3条带型清晰、重复性好的ISSR引物,通过对PCR扩增及电泳得到的DNA片段条带进行聚类分析并构建聚类树状图,并对陆川猪种群进行遗传差异分析。结果显示,ISSR分子标记上共发现182条条带,其中84条多态性条带,3条ISSR引物扩增出来的总DNA片段条带与多态性DNA片段条带差异较大且分布不均匀,表明所选择的3个ISSR分子标记在陆川猪种群中具有丰富的多态性;陆川猪种群之间遗传相似系数变异范围为0.43~1.00,表明种群间的遗传差异变化较大,遗传背景丰富;UPGMA聚类分析表明,陆川猪与长陆二元杂猪、巴马香猪、环江香猪均可聚为一类,大陆二元杂猪、三元杂猪和龙宝猪聚为一类。本研究证实了所选的3条ISSR引物可作为有效的遗传标记用于陆川猪种群间的遗传差异分析,丰富了当前的陆川猪ISSR分子标记资源,同时也表明陆川猪种群具有丰富的遗传多样性。     

鸽痘病毒DNA限制性内切酶分析

利用蔗糖密度梯度离心纯化鸽痘病毒 ,以蛋白酶 K法抽提病毒 DNA,提取到高分子量病毒 DN、病毒 DN　经 Eco R 、Sal ,Pst 酶切后各产生 17、8和 13个片段 ,病毒 DNA的大小约为 2 89.32 kb。     

Bovine papillomavirus DNA in neoplastic and nonneoplastic tissues obtained from horses with and without sarcoids in the western United States

OBJECTIVE: To determine the incidence of bovine papillomavirus (BPV) type 1 or 2 in sarcoids and other samples of cutaneous tissues collected from horses in the western United States. ANIMALS: 55 horses with sarcoids and 12 horses without sarcoids. PROCEDURE: Tissue samples (tumor and normal skin from horses with sarcoids and normal skin, papillomas, and nonsarcoid cutaneous neoplasms from horses without sarcoids) were collected. Tissue samples were analyzed for BPV-1 or -2 DNA, using a polymerase chain reaction (PCR) and restriction fragment length polymorphism. The PCR products from 7 sarcoid-affected horses were sequenced to evaluate percentage homology with expected sequences for BPV-1 or-2. RESULTS: Most (94/96, 98%) sarcoids contained BPV DNA. Sixty-two percent of the tumors examined had restriction enzyme patterns consistent with BPV-2. Thirty-one of 49 (63%) samples of normal skin obtained from horses with sarcoids contained BPV DNA. All samples subsequently sequenced had 100% homology with the expected sequences for the specific viral type. All tissues from healthy horses, nonsarcoid neoplasms, and papillomas were negative for BPV DNA. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine papillomaviral DNA was detected in essentially all sarcoids examined. There appears to be regional variation in the prevalence of viral types in these tumors. The fact that we detected viral DNA in normal skin samples from horses with sarcoids suggests the possibility of a latent viral phase. Viral latency may be 1 explanation for the high rate of recurrence following surgical excision of sarcoids.     

Molecular carcinogenesis of canine mammary tumors: news from an old disease

Studies focusing on the molecular basis of canine mammary tumors (CMT) have long been hampered by limited numbers of molecular tools specific to the canine species. The lack of molecular information for CMT has impeded the identification of clinically relevant tumor markers beyond histopathology and the introduction of new therapeutic concepts. Additionally, the potential use for the dog as a model for human breast cancer is debatable until questions are answered regarding cellular origin, mechanisms, and cellular pathways. During the past years, increasing numbers of canine molecular tools have been developed on the genomic, RNA, and protein levels, and an increasing number of studies have shed light on specific aspects of canine carcinogenesis, particularly of the mammary gland. This review summarizes current knowledge on the molecular carcinogenesis of CMT, including the role of specific oncogenes, tumor suppressors, regulators of apoptosis and DNA repair, proliferation indices, adhesion molecules, circulating tumor cells, and mediators of angiogenesis in CMT progression and clinical behavior. Whereas the data available are far from complete, knowledge of molecular pathways has a significant potential to complement and refine the current diagnostic and therapeutic approach to this tumor type. Furthermore, current data show that significant similarities and differences exist between canine and human mammary tumors at the molecular level. Clearly, this is only the beginning of an understanding of the molecular mechanisms of CMT and their application in clinical patient management.     

Ultrasound-guided serial transabdominal cardiac biopsies in cats

A percutaneous/transdiaphragmatic core needle biopsy technique was developed in cats to obtain serial biopsies from different locations of the left ventricle, through which morphological and molecular changes within the same individual can be studied to unravel the mechanisms of feline cardiomyopathies. Transmural left ventricular myocardial samples were obtained from 29 anesthetized, healthy, adult cats with ultrasound guidance. An 18G automatic biopsy needle was inserted between the last left rib and the sternum through the diaphragm into the thorax. Biopsies were obtained from the left ventricular wall. In five cats, three single biopsies were taken with 4-week intervals. Autopsy was performed on six cats, of which three cats had serial biopsies. In total, 87 biopsies were obtained without long-term effects on cardiac function or structure. The biopsies caused transient single ventricular premature complexes and mild pericardial effusion without tamponade. Necropsy revealed a minimal amount of fibrous connective tissue in the diaphragm and the heart without any significant microscopic lesions in the adjacent muscle tissue. The high quality biopsy material was suitable for morphological and molecular studies. This minimally invasive, ultrasound-guided cardiac biopsy technique thus allows for the safe collection of serial biopsies to study feline cardiomyopathies in an experimental setting.     

Identification of parvovirus in the bone marrow of eight cats

Objective To determine if canine parvovirus (CPV) or feline panleucopenia virus (FPV) genomic sequences are present in adult feline bone marrow samples. Design Bone marrow samples were obtained from 32 semi‐feral cats that were euthanased at an animal shelter. DNA was extracted and subjected to conventional polymerase chain reaction (PCR) designed to determine if CPV or FPV DNA was present. Positive PCR products were purified, cloned and sequenced to differentiate between CPV and FPV. Results Eight of the bone marrow samples contained parvoviral DNA (7 CPV, 1 FPV). Conclusion CPV and FPV DNA can be found in the bone marrow of healthy adult cats.     

Detection of ovine intramammary infection with the California mastitis test

The overall sensitivity of the California mastitis test (CMT) for detecting intramammary infection (the likelihood of a positive CMT score in the presence of intramammary infection) was 69.3%. The specificity of the CMT (likelihood of a negative test in the absence of intramammary infection) was 76.5%. When only infections by major ovine mastitis pathogens were considered, the sensitivity increased to 100% and the specificity decreased to 71.1%. Bacterial cultural examination and CMT scores were recorded for 526 samples of milk obtained from 106 brood ewes. Positive CMT scores were recorded for all samples from udder halves infected with major ovine mastitis pathogens (coagulase-positive staphylococci, Escherichia coli, and Pasteurella haemolytica). The CMT scores for samples from udder halves infected with coagulase-negative staphylococci were variable.     

一种高效提取桑树根围土壤微生物总DNA的方法

为了建立桑树根围土壤微生物群落结构的分子生态学试验技术体系,采取不同的细胞裂解方法及DNA沉淀方法直接提取桑树根围土壤微生物总DNA,并以采用试剂盒提取的桑树根围土壤微生物总DNA为参照,筛选出一种能有效提取高纯度、多样性土壤微生物总DNA的方法:以十六烷基三甲基溴化铵(CTAB)、溶菌酶、蛋白酶K、十二烷基磺酸钠(SDS)为裂解液,经反复冻融裂解细胞,在DNA提取液中添加聚乙烯吡咯烷酮K30(PVP K30)、牛血清蛋白(BSA)、CaCl2去除腐殖酸,并结合聚乙二醇8000(PEG8000)沉淀和纯化DNA。该方法简便、快速,获得的桑树根围土壤微生物基因组DNA分子长度在23 kb以上,土壤鲜样的微生物DNA产率为11.2μg/g,且纯度较高,D(260 nm)/D(230 nm)=1.42,D(260 nm)/D(280 nm)=1.37,可直接用于PCR扩增及变性梯度凝胶电泳(DGGE)分析。     

鸡呼吸道传染病基因芯片诊断方法的建立

随着养禽业集约化程度的不断提高,鸡呼吸系统疾病的发生呈逐年上升趋势.而且大多数的呼吸道疾病不是单一病原感染,而是多种病原混合感染,从临床上难以及时鉴别诊断,延误防治时机,从而给养禽业造成巨大的经济损失.因此,禽呼吸道疾病已经成为生产和研究中的一类重要疾病.目前,禽呼吸系统疾病的诊断方法主要有病毒分离、血凝（HA）、血凝抑制（HI）、琼脂扩散、ELISA、PCR等,但是传统的检测方法灵敏度较低,而且当病原发生变异或感染禽处在潜伏期时会造成误诊.PCR方法每次只能检测一种病毒,费时费力.采用基因芯片的方法可以快速、准确地同步检测多种病原体,且需要样品量少、灵敏、特异、快速、费用低廉,将生物芯片技术用于禽呼吸道疾病诊断意义重大.     

淮阴苜蓿SSR指纹图谱的构建

采用不同的取样策略,利用SSR分子标记研究淮阴苜蓿（Medicago sativa Huaiyin）遗传多样性,确定最佳取样数,在此基础上利用筛选的SSR引物构建淮阴苜蓿的指纹图谱,并对淮阴苜蓿进行品种鉴定。本研究对320株淮阴苜蓿的单株基因组DNA设置6个处理（10、20、30、40、50、60单株DNA随机等量混合）,每处理8个重复,利用筛选的4对SSR引物进行遗传多样性分析。结果表明,40株单株随机DNA的混合样品的遗传多样性指数开始趋于稳定,由此取40株即可代表淮阴苜蓿群体。采用取样数为40株单株DNA混合样,利用筛选的3对SSR引物构建淮阴苜蓿指纹图谱,并对15份供试苜蓿材料进行检测,结果表明,该方法所构建淮阴苜蓿SSR指纹图谱可以用于对淮阴苜蓿品种的鉴定。     

采用改良CTAB法提取柞树(Quercus L.)基因组DNA

为了获得质量较高的柞树基因组DNA,采用经过改良的十六烷基三乙基溴化铵(CTAB)提取法对几种常见柞树植物进行基因组DNA的提取,所得到的DNA样品经紫外分光光度计和琼脂糖凝胶电泳检测,纯度较高,D260 nm/D280 nm为1.75~1.80,应用于SSR和RAPD标记分析时,可以获得较好的扩增片段。     

Identification of Helicobacter DNA in feline pancreas, liver, stomach, and duodenum: comparison between findings in fresh and formalin-fixed paraffin-embedded tissue samples

The clinical significance of Helicobacter spp. in feline digestive organs needs to be evaluated and formalin-fixed and paraffin-embedded (FFPE) tissue samples provide an invaluable source for molecular studies. In this study, we performed a PCR assay to investigate the presence of Helicobacter DNA in digestive organs from seven cats and compared this occurrence in fresh and formalin-fixed and paraffin-embedded (FFPE) tissue samples from the same organs. The present study identified Helicobacter DNA in the pancreas, liver, stomach, and duodenum in fresh tissue samples but only in the stomach in FFPE samples. To our knowledge this is the first time that Helicobacter DNA have been identified in the feline pancreas. This study indicates that it is important to be aware of differences between results when analyzing FFPE samples compared to fresh tissue samples, especially regarding longer DNA fragments (>200 bp (base pairs)).