不同部位取样对鸡基因组DNA提取的效果的影响

采用伤害性取样和非伤害性取样,采集鸡的肝脏、血液、羽髓、羽鞘、羽根、实心羽茎和羽毛上脐周围绒羽等不同部位样品,并采取对样品不同处理程序提取基因组DNA。结果表明,鸡的微量血液(50μl)和羽髓样品提取基因组DNA与肝脏(50 mg)样品提取的基因组DNA的量相当;羽鞘、羽根、实心羽茎和羽毛上脐周围绒羽可以提出一定量的基因组DNA,其提取量少于肝脏和羽髓组织,但可以满足各种涉及PCR反应的分子生物学试验,肝脏羽髓消化最适时间为2.5 h,其他样品需消化过夜。该研究结果为禽类分子生物学研究拓宽了样本来源。     

褐马鸡非损伤性取样、微量取样DNA提取的初步研究

褐马鸡是中国特有的国家Ⅰ级保护濒危鸟类,为了解决野生褐马鸡分子生物学研究中存在的取样局限性,采用非伤害性取样和非损伤性取样,对褐马鸡微量血液、皮、脚垫、指甲、羽毛、陈旧标本的皮以及粪便的DNA提取进行了初步研究。结果表明,从褐马鸡微量血液（10μL）中可以提取到浓度和纯度高的基因组DNA（41.85μg）;从褐马鸡的皮、脚垫、指甲、羽毛中可以提取出基因组DNA并扩增出目标片断;陈旧标本的皮能够提取到基因组DNA。该研究将拓宽褐马鸡分子生物学研究的采样范围,并可以提高野外非损伤性取样在保护遗传学中的应用。     

非损伤性取样提取动物DNA方法探讨

近年来,随着分子生物学技术的发展,非损伤性取样逐渐成为野生动物分子生物学研究中获取试验材料的主要方法.文章主要通过介绍当前非损伤取样提取动物DNA的方法,比较各种方法的优缺点和局限性,并分析了各种方法的研究特点和应用前景,以期对下一步研究提供有价值的参考.     

羽髓细胞短期孵育制备染色体标本的研究

国内目前有关鸟类染色体制备方法的研究报道较多 ,但其中大多数方法都对鸟类有不同程度的损伤或实验技术条件要求过高 ,不利于推广利用。本文报道的羽髓细胞短期孵育制备鸟类染色体标本的方法 ,是以鸟类雏鸟羽毛的羽髓或成鸟换羽期新长出的飞羽羽髓为实验材料 ,经含有秋水仙素的培养基短期孵育 ,然后采用常规制片方法制备染色体标本。结果表明 ,此方法取材容易 ,操作简便 ,对鸟类无损伤 ,是适用于濒危鸟类的细胞遗传学研究的理想方法。     

一种改进的方法提取白头鹤粪便DNA

现有的粪便DNA提取方法通用性不好,DNA纯度不高不适用于白头鹤等水鸟,本研究探索一种改进的硫氰酸胍裂解法。通过线粒体DNA细胞色素b、控制区基因和微卫星DNA的PCR扩增及线粒体DNA控制区序列的测定,经琼脂糖凝胶电泳检测以及测定序列显示,提取的DNA模板纯度高,效果很好,证实改进方法的可靠性、有效性。为其他濒危水鸟的非损伤取样提供了新的材料和方法,为其保护遗传学的研究提供一定的新技术。     

双向电泳分析鸡羽髓蛋白组学方法的建立

为建立双向电泳方法研究鸡羽髓蛋白组学,以进一步了解病毒与宿主相互作用的新机制,从SPF鸡羽根挤出羽髓,提取其中蛋白进行双向电泳,并对不同裂解液、IPG胶条pH值范围、一向等电聚焦条件、二向SDS-PAGE凝胶浓度等影响因素进行优化.结果显示,采用17cm,pH 5～8 1PG胶条,400μg羽髓蛋白进行的双向电泳,图谱用PDQuest8.0.1分析,可分辨的蛋白点约为700左右,不同样本间的匹配率大于80%.可见,本研究建立的双向电泳方法分辨率及重复性均较高.     

六种试剂盒提取血液基因组DNA效果比较

为筛选一种高效、快捷的血液基因组DNA提取试剂盒,采集24份新鲜西藏绵羊血液样本,选用6种市售商业化试剂盒（LifeFeng柱式试剂盒DK601和非柱式试剂盒DK602,康为世纪非柱式试剂盒CW0544和柱式试剂盒CW0546,上海生工非柱式试剂盒SK8224和柱式试剂盒SK8254）,比较所提DNA的浓度、纯度、得率以及对嗜吞噬细胞无形体16S rRNA进行巢式PCR扩增,综合评价不同试剂盒DNA提取效果.结果表明,应用LifeFeng柱式试剂盒DK601提取的DNA浓度和纯度均最高,得率较高,分别达到75.63 ng/μL、1.894（OD260/OD280）、18.91 ng/μL;且嗜吞噬细胞无形体16S rRNA基因的PCR扩增条带清晰度最优.因此,LifeFeng DK601可作为提取绵羊血液基因组DNA及嗜吞噬细胞无形体（Anaplasma phagocytophilum）病原鉴定的首选试剂盒.     

从白羽王鸽的血液中提取基因组DNA方法的比较研究

禽类血液中的蛋白质含量较高,用传统方法提取其血液中的基因组DNA比较繁琐、纯度不高,而且提取的产物中有大量蛋白质残留。本研究采用改良的CTAB法从白羽王鸽全血中提取全基因组DNA,并以传统的酚-氯仿抽提的方法和TAKARA公司基因组提取试剂盒法作对照。结果表明：qCTAB法可以得到大量的、较完整的基因组DNA,并且纯度达到2．00,浓度达到了144．67ng/μL,完全可以满足PCR扩增限制酶切等实验的要求。     

全血中DNA的5种不同提取方法比较研究

比较几种从血液中提取DNA方法所需要的时间、样本量、提取的DNA纯度、产量以及成本等。结果表明,几种方法所提取的DNA质量都能达到分子生物学的试验要求,但在DNA的纯度、产量、试验时间和价格等方面存在差别。实验人员可根据自己的实验要求、实验室以及经济条件等选择适当的方法。     

对猪肝脏基因组DNA提取与纯化的研究

对血液中提取基因组DNA的方法加以改进,获得了一种提取猪肾组织中DNA的有效方法.试验表明,利用SDS裂解,氯仿、异戊醇抽提母猪肝脏中基因组DNA,得到的DNA纯度较高,可用来进一步进行RAPD分析和PCR扩增,用于各种分子生物学实验.     

Isolation of genomic DNA from feathers.

The use of feathers in veterinary clinical practice simplifies the sampling of avian genomic DNA, especially when blood extraction is difficult because of the age or the size of the bird. A rapid and accurate protocol was used to isolate high-quality genomic DNA from feathers. The technique includes a lysis step of the feather quill, which differs in temperature and time of incubation depending on the feather size. Purification of genomic DNA is performed with phenol: chloroform: isoamyl alcohol extraction and ethanol precipitation. This protocol consistently provided significant amounts of high-quality genomic DNA from more than 800 birds belonging to 120 different species. Genomic DNA isolated with this method was used for Southern blotting and also in several polymerase chain reaction systems devoted to sex determination and paternity testing.     

利用羽髓组织分析濒危鸟类同工酶

鸟类，特别是濒危鸟类同工酶的分析，长期以来一直受到组织学样品来源上的限制。因为在分析实验中普遍采用血液和各种新鲜组织为分析材料，这常常要损伤或杀死动物，对濒危鸟类的保护是不利的。本文以生长羽毛的羽髓组织为材料，进行了乳酸脱氢酶（ＬＤＨ）同工酶的凝胶电泳分析。所得谱带清晰，重复性好，且对鸟体无伤害，是濒危鸟类同工酶分析的理想方法。     

Polymerase chain reaction for detection of avian leukosis virus subgroup J in feather pulp

Feather pulp from experimentally infected chickens was used as a source of DNA for polymerase chain reaction (PCR) amplification of avian leukosis virus subgroup J (ALV-J) proviral DNA. A primer set that produces a large amplicon (approximately 2,125) was used to detect ALV-J proviral DNA. This primer set was used in lieu of previously published primers because it allows for sequencing of the entire envelope gene and because it was able to detect diagnostically a number of North American ALV-J isolates that could not be detected with previously published primers and PCR conditions. ALV-J proviral DNA was detected in feather pulp at 7 days of age in more than 90% of birds infected as embryos and 7 days postinoculation in over 50% of chickens infected at 3 days of age. The results obtained with PCR on feather pulp were compared with those of virus isolation. In the embryo-inoculated birds, the percentages of agreement between PCR and virus isolation were 92.5% at 7 days of age and 100% at 28, 42, 49, and 56 days of age. However, the overall sensitivity of virus isolation in embryo-infected birds was higher, particularly at 7 and 56 days of age. In chickens inoculated at 3 days of age, the percentages of agreement of detection between PCR and virus isolation ranged from 75% at 10 days of age to 100% at 42 days of age. Agreement of negative results of ALV-J detection by PCR and virus isolation in chickens infected posthatch ranged between 66.6% and 100% between the ages of 10 and 42 days. Virus isolation requires chicken embryo fibroblasts of specific genetic lines, and the process takes onaverage 7-9 days. Aseptic collection of blood and tissues for virus isolation and molecular detection of ALV-J requires sterile necropsy instruments as well as syringes and needles for each individual chicken, whereas sterile microcentrifuge tubes and gloves are the only equipment necessary for aseptic feather pulp collection for ALV-J detection by PCR. PCR-based detection of ALV-J in feather pulp is especially suitable when ALV-J infection must be diagnosed rapidly and unequivocally without killing the chicken(s) and in situations where crucial reagents or suitable virus propagation substrates are not readily available for isolation and propagation of ALV-J in cell culture.     

Comparison of Newcastle disease vaccines by serology using serum,tears and feather pulp samples

Seroconversion of 3 lentogenic commercial Newcastle disease (ND) vaccines and experimental V₄ vaccines was compared based on the haemagglutination inhibition (HI) test against ND. It was found that for primary vaccination all the vaccines produced similar response but for secondary vaccinations V₄ and LaSota were better than RDVF. Eighty-five samples each of serum, tears and feather pulp were collected from respective birds and antibody assessment was done against ND by HI test. The geometric mean HI titres (GMT) of serum samples were highest followed by tears and feather pulp samples before vaccination and 3 weeks after vaccination by oculonasal route and the difference was statistically significant (p<0.01). Three weeks after booster vaccination by oculonasal route, however, the GMT of serum samples were highest followed by feather pulp and tears samples. The ease of collection of feather pulp samples and their role in ND serology is discussed.     

Comparison of blood and feather pulp samples for the diagnosis of Marek's disease and for monitoring Marek's disease vaccination by real time-PCR

Comparison of blood and feather pulp (FP) samples for the diagnosis of Marek's disease (MD) and for monitoring Marek's diseases vaccination in chickens (serotypes 2 and 3 vaccines) by real time-PCR was evaluated. For diagnosis of MD, quantification of serotype 1 Marek's disease virus (MDV) DNA load was evaluated in 21 chickens suffering from MD. For each chicken, samples of blood and FP were collected and MDV DNA load was quantified. Solid tumors are the sample of choice for MD diagnosis by real time-PCR and, hence, 14 solid tumors were included in the study as positive controls. Load of MDV DNA in FP was equivalent to that detected in solid tumors (threshold cycle [Ct] ratio above 1.7). MDV DNA load in blood samples was lower than in solid tumors and FP samples. Nonetheless, there was a statistically significant correlation of the results obtained from FP and blood (r = 0.92). Results of the Pearson correlation test showed that Ct ratio values of 1.7 in FP correspond to Ct ratio values of 1.2 in peripheral blood. For monitoring vaccines, serotypes 2 and 3 MDV DNA load was evaluated in blood and FP samples of vaccinated chickens. Serotype 2 MDV DNA load was evaluated in samples of blood and FP from 34 chickens vaccinated with SB-1 strain. Serotype 3 MDV DNA load was evaluated in blood and FP samples from 53 chickens vaccinated with HVT strain. For both serotypes, frequency of positive samples and load of vaccine DNA was higher in FP than in blood samples. There was not a statistically significant correlation between the load of SB-1 DNA (r = 0.17) or HVT DNA (r = -0.04) in FP and blood. Our results show that the load of serotypes 1, 2, and 3 DNA is higher in FP than in blood. Diagnosis of MD could be done using both FP and blood samples. Monitoring of MD vaccination by real time-PCR required the use of FP samples. There were a high percentage of false negative samples when using blood to detect serotypes 2 and 3 MDV by real time-PCR.     

High virus titer in feather pulp of chickens infected with subgroup J avian leukosis virus

Subgroup J avian leukosis viruses (ALVs), which are a recombinant virus between exogenous and endogenous ALVs, can spread by either vertical or horizontal transmission. Exogenous and endogenous ALVs can be detected in feather pulp. In this study, virus titers in feather pulp of chickens infected with subgroup J ALV were compared with those of plasma and cloacal swab. All of the broiler chickens inoculated with subgroup J ALV at 1 day old were positive for virus from feather pulp during the experimental period of between 2 wk and 8 wk of age. Virus titers in feather pulp of some broiler chickens infected with subgroup J ALV were very high, ranging from 10(7) to 10(8) infective units per 0.2 ml. Virus titers in feather pulp were usually the highest among the samples of plasma, cloacal swab, and feather pulp tested. In another experiment in which layer chickens were inoculated with subgroup J ALV at 1 day old, virus was detected in feather pulp from 2 wk until 18 wk of age, and virus persisted longer in feather pulp than in plasma. Almost all of the layer chickens tested were positive for virus by polymerase chain reaction (PCR) with DNA extracted from feather pulp samples at 2, 4, and 10 wk of age, and the PCR from feather pulp was more sensitive than virus isolation from plasma, cloacal swab, and feather pulp. All above results indicate that samples of feather pulp can be useful for virus isolation and PCR to confirm subgroup J ALV infection.     

一例朱鹮微量元素锌缺乏性疾病的诊治

2008年4月,陕西汉中朱鹮国家级自然保护区人工饲养的1只朱鹮背部羽毛严重缺失,病变部位绒羽脱落,片羽羽干质脆易断,中下部羽丝缺失,在距皮肤0.3～0.5cm处折断脱落。实验室诊断发现血液中微量元素锌含量较低,通过补充微量元素锌进行治疗,取得了较好的治疗效果。     

A survey of avian polyomavirus (APV) infection in imported and domestic bred psittacine birds in Japan

Although birds infected with avian polyomavirus (APV) subclinically could be a source of infection, no epidemiological studies of APV in psittacine birds have been reported in Japan. In the present study, we investigated subclinical morbidity rate of APV in imported and domestically bred psittacine birds by polymerase chain reaction (PCR). Of 402 live birds from which blood or feather samples were taken between April, 2003 and March, 2004, 11 (2.7%) were found to be APV positive. The DNA sequences of the APV t/T antigen region were determined for five APV-positive randomly selected samples and were found to be conserved.     

The presence of quill mites (Gabucinia bicaudata) and lice (Struthiolipeurus struthionis) in ostrich wing feathers

Quill mites (Gabucinia bicaudata) and lice (Struthiolipeurus struthionis) may infest ostrich feathers, resulting in skin damage, pruritus and excessive feather preening and loss. Four different feather types (prime white, femina extra wide, femina class 1, and femina short; n = 10) were collected. The quill mites and lice were removed with fine forceps, studied using a photographic optical microscope and counted microscopically at x 100 magnification following collection by sedimentation. They were placed in separate Petri dishes containing lactophenol solution and examined (x40 magnification). Anatomical features are described. The density of quill mites in all feather types of both wings was higher than that of the lice. There was no significant difference between the counts of both arthropods on the left wing and the right wing, respectively, except for the femina class 1 quill mites (P = 0.01). The femina extra wide feathers were a preferred habitat in both wings. Large standard deviations (quill mites left wing: 73 +/- 8; quill mites right wing: 69 +/- 7) suggested variations in the degree of migration between feather shafts or as a response to escape preening. It is recommended that ostriches be treated with an oral preparation of Ivermectin administered per os at a dosage rate of 0.2 mg/kg at 30-day intervals for quill mites, and with a 1-5 % Malathion dust at 14-day intervals for lice.