PCR-DGGE实验技术在畜牧科学研究中的应用

本文综述了以变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)为主要手段结合聚合酶链式反应(polymerase chain reaction,PCR)实验技术的基本原理、主要实验流程,优缺点以及它在畜禽肠道等微生物的群落结构和多样性研究的最新动态。     

DGGE技市在动物胃肠道微生态研究中的应用

变性梯度凝胶电(Denaturing Gradient Gel Electrophoresis,DGGE)术是通过直接分离核酸片段对微生物群落进行研究,由于其具有可靠性强、重复性好、方便快捷等优点.被广泛应用于微生物群落多样性和动态性分析,并成为微生物分子生态学研究中的热点技术之一.本文主要介绍DGGE技术的原理、工作流程及优缺点.并着重分析了其在动物胃肠道微生态研究中的应用.     

DGGE技术及其在瘤胃微生物多样性研究中的应用

基于可靠性强、重现性高、方便快捷等优点,变性梯度凝胶电泳（DGGE）技术已经成为微生物群落多样性和动态分析的强有力工具。本文在阐述PCR—DGGE原理及流程的的基础上,对DGGE技术在瘤胃微生物多样性研究中的具体应用及其局限性与改进方法进行了全面阐述。     

变性梯度凝胶电泳（DGGE）的研究进展

DGGE技术是由Fischer等于1979年提出的一种用于检测DNA突变的电泳技术[1],之后Myers等首次在DGGE的引物中使用“GC夹子”,使得突变检出率大大提高,从而进一步完善了该技术。1993年,Muyzer等将DGGE技术应用于微生物的生态学研究,并且证实了该技术在研究自然界微生物群落的种群差异性和遗传多样性方面具有突出的优越性[2]。与传统的菌种分离培养技术及生理生化指标检测相比,DGGE技术通过对微生物群落的核酸信息进行分析,能更准确、更快速的对菌群进行鉴定,同时可鉴定出自然状态下不可培养的菌株。由于DGGE技术具有重现性高、可靠性强和高效快捷等优点[3],现以用于微生物群落的复杂性分析、检测微生物种群动态变化、对比细菌的富集培养及分离培养、单基因组中rRNA的多样性分析、DNA提取方法比较等方面,在废水、海洋、森林、土壤等环境样品研究以及发酵工艺、植物内生真菌研究、种群演替规律研究等领域广泛应用。     

变性梯度凝胶电泳(DGGE)的研究进展

DGGE技术是由Fischer等于1979年提出的一种用于检测DNA突变的电泳技术[1],之后Myers等首次在DGGE的引物中使用"GC夹子",使得突变检出率大大提高,从而进一步完善了该技术。1993年,Muyzer等将DGGE技术应用于微生物的生态学研究,并且证实了该技术在研究自然界微生物群落的种群差异性和遗传多样性方面具有突出的优越性[2]。与传统的菌种分离培养技术及生理生化指标检测相比,DGGE技术通过对微生物群落的核酸信息进行分析,能更准确、更快速的对菌群进行鉴定,同时可鉴定出自然状态下不可培养的菌株。由于DGGE技术具有重现性高、可靠性强和高效快捷等优点[3],现以用于微生物群落的复杂性分析、检测微生物种群动态变化、对比细菌的富集培养及分离培养、单基因组中r RNA的多样性分析、DNA提取方法比较等方面,在废水、海洋、森林、土壤等环境样品研究以及发酵工艺、植物内生真菌研究、种群演替规律研究等领域广泛应用。     

瘤胃微生物生态研究方法评述

瘤胃微生物生态研究方法主要经历了微生物纯培养、混合培养、微生物分子生物学技术[(从瘤胃中提取DNA,进行PCR-16S rDNA(rRNA)技术]、变性梯度凝胶电泳(DGGE)技术),在不同程度上揭示了瘤胃微生物群落丰富的多样性和生态功能.但由于各种方法本身的局限性,限制了人类对瘤胃微生物的全面了解,现代生物技术和传统微生物研究方法的配合将为瘤胃微生物生态研究提供较好的前景.     

应用PCR-DGGE分析肉鸡呼吸道中细菌多样性

从不同养殖时期的肉鸡呼吸道中提取微生物基因组总DNA,以细菌16S rDNA基因通用引物341F/534R进行V3高变异区域PCR扩增,长约200 bp的PCR产物经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的特征DNA指纹图谱。通过DGGE图谱的半定量分析以及对条带的测序鉴定,发现样品的优势菌群明显。结果表明,PCR-DGGE是研究动物呼吸道微生物多样性的可行方法。     

瘤胃微生物定量方法的研究进展

当前人们对瘤胃中微生物的认识只有10%～20%,不断改进研究技术和手段,才能大大推动瘤胃微生态领域的研究。作者阐述了瘤胃微生物传统定量方法（滚管计数法和最大或然数法）和分子生物学定量方法,如探针杂交技术、荧光原位杂交、DGGE、定量PCR和流式细胞计量术（flow cytometry )等的应用状况及其各自的优缺点。     

应用PCR-DGGE分析南美白对虾肠道微生物多样性

从南美白对虾肠道中提取微生物基因组总DNA,以细菌16SrRNA基因通用引物341F/534R进行V3高变异区域PCR扩增,长约200bp的PCR产物纯化后经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的特征DNA指纹图谱。通过DGGE图谱的半定量分析,发现样品的优势群落明显。结果表明,PCR-DGGE是研究水产动物肠道微生物多样性的可行方法。     

PCR-DGGE技术及其在微生物群落结构研究中的应用

PCR-变性梯度凝胶电泳（PCR—DGGE）技术具有可靠性强、重复性好、方便快捷等优点，是一种被广泛应用于环境生态学中微生物群落多样性、动态性分析和功能细菌跟踪的指纹识别技术。本文介绍了PCR—DGGE分析技术的基本原理和方法，并就该方法在对生态环境中、动物和人体内微生物群落结构的研究应用情况做了综述，并对该技术自身存在的局限性和应用前景进行了评价。     

The microflora and concentrations of volatile fatty acids in the rumen of cattle fed on single component rations

The composition of the rumen microflora and the volatile fatty acids were examined in cattle free-grazing on grass or stall-fed on hay, grass pellets, oats or dried beet pulp with molasses. Total and viable counts of anaerobic bacteria were highest on the grass feeding, but viable counts as a percentage of total counts were highest when oats or beet pulp with molasses were fed. Counts of cellulolytic bacteria were lowest on these latter 2 diets, and highest on grass or grass pellet diets. Studies of the anaerobic flora showed that the composition in animals fed on grass pellets resembled more that found in animals free-grazing on grass than in those fed on hay. Counts of aerotolerant bacteria were only a small percentage of the total count, but were highest on the hay diet. On this latter diet and on grass-feeding the streptococci (identified as Streptococcus bovis) were predominant, but contrary to expectation, streptococci were found only in small numbers on the oats diet, where coryneform rods were the major type present. Although a period of 4–6 weeks was allowed for the animals to adapt to the feeds, the 2 periods of feeding on oats and dried beet pulp with molasses markedly affected the composition of the rumen flora in the subsequent periods of feeding grass pellets and hay. Ruinen volatile fatty acid analysis showed a propionogenic effect of oats and the highest percentage of butyric acid when beet pulp with molasses was fed. The expected propionogenic effect of grass pellets was not observed.     

米糠多糖对健康雏鸡外周血T淋巴细胞转化和体液免疫的影响

为探讨米糠多糖对鸡免疫功能的影响,选用100只10日龄京白939蛋雏鸡,随机平均分为4组。Ⅱ、Ⅲ、Ⅳ组从10日龄起分别灌服低、中、高3个剂量米糠多糖(50、100和200mg/(kg.d)),每天一次性灌服规定剂量,连续5d;Ⅰ组同时灌服生理盐水作为对照,1mL/只。在灌服米糠多糖前1d和灌服后7、14和21d,分别测定雏鸡外周血T淋巴细胞转化能力和血清IgG、IgA和IgM含量。在雏鸡14日龄时,对照组和米糠多糖处理组均实施新城疫(ND)疫苗免疫,间隔1周后进行禽流感(AI)疫苗免疫。分别在ND疫苗和AI疫苗免疫前1d和免疫后7、14和21d测定ND抗体和AI抗体效价。结果显示:(1)雏鸡灌服3个不同剂量米糠多糖后的7、14和21d,外周血T淋巴细胞转化能力均显著高于对照组(P<0.05或P<0.01),且以100mg/(kg.d)剂量组效果最好。(2)雏鸡服用3个剂量组米糠多糖后7、14和21d,血清IgG含量均高于对照组(P<0.05或P<0.01);血清中IgA含量,在14d时,低剂量组与对照组无显著差异,中、高剂量组显著高于对照组(P<0.05或P<0.01);IgM含量在灌服米糠多糖后7d时,Ⅳ组高与对照组无差异(P>0.05);14d时,Ⅲ组与对照组无差异(P>0.05);米糠多糖其他剂量组在7和14d,血清IgM含量均高于对照组(P<0.05或P<0.01);21d时,米糠多糖处理组与对照组比较无显著差异。(3)雏鸡服用米糠多糖可以提高ND疫苗和AI疫苗免疫后的特异性抗体效价,且免疫效果与米糠多糖剂量呈正相关。试验结果表明,米糠多糖能够从促进蛋雏鸡外周血T淋巴细胞转化功能、提高血清免疫球蛋白含量以及提高疫苗免疫特异性抗体效价等方面调节健康雏鸡的免疫功能。     

甘肃省草本植物上几种黑粉病及病原鉴定

对采自甘肃的画眉草黑粉病 (Ustilago sp ermophora)、白羊草黑粉病 (Sporisoriumandropogonis)、草玉梅黑粉病 (Urocystis anemones)和蓼黑粉病 (Microbotryum bistortarum)的症状进行了描述 ,并对病原进行了鉴定、光学和电镜观察。这 4种病原及引起的病害在甘肃省以前未见正式报道     

内蒙古典型草原野生草坪草种的收集和栽培试验

试验对内蒙古典型草原进行了草坪草种质资源调查,经过野外观察采集了20种草种,并进行了盆栽和建坪试验。结果发现,很多野生的草种都是很好的草坪草材料,如苔草属(Carex)和塔草属(Koeleria)的一些草种表现出巨大的潜力。最后提出了我国草坪草种质资源调查和育种工作的一些建议。     

USE OF THE T2*‐WEIGHTED GRADIENT RECALLED ECHO SEQUENCE FOR MAGNETIC RESONANCE IMAGING OF THE CANINE AND FELINE BRAIN

T2*‐weighted magnetic resonance imaging (MRI) has been reported to help improve detection of intracranial hemorrhage and is widely used in human neuroimaging. To assess the utility of this technique in small animals, interpretations based on this sequence were compared with those based on paired T2‐weighted and fluid‐attenuated inversion recovery (FLAIR) sequences in 200 dogs and cats that underwent brain MRI for suspected intracranial disease. Two sets of images (T2 + FLAIR and T2*) were reviewed separately in random order unaccompanied by patient information and were interpreted as normal or abnormal based on whether intracranial abnormalities were seen. The number and location of intracranial lesions were recorded. Eighty‐five studies were considered normal and 88 were considered abnormal based on both sets of images, with good agreement (κ = 0.731) between the two. Susceptibility artifact was present in 33 cases (16.5%) on T2*‐weighted images. In 12 cases (6%) a total of 69 lesions were seen on T2*‐weighted images that were not seen on T2/FLAIR, all of which were associated with susceptibility artifact caused by presumed intracranial hemorrhage. Pseudolesions were seen on T2*‐weighted images in five cases, none of which were associated with susceptibility artifact. Abnormalities were seen on T2/FLAIR images that were not seen on T2*‐weighted images in 35 cases, confirming that T2* does not replace standard spin echo sequences. These results support inclusion of T2*‐weighted sequences in small animal brain MRI studies and indicate that that a large number of abnormalities (especially hemorrhagic lesions) can go undetected if it is not performed.     

Evaluation of 18F‐FDG PET/CT as a diagnostic imaging and staging tool for feline oral squamous cell carcinoma

18F‐fluorodeoxyglucose positron emission tomography combined with computed tomography (18FDG‐PET/CT) has been shown to be effective for staging human oral squamous cell carcinoma (SCC) but its application for cats with oral SCC is unknown. Twelve cats with biopsy‐proven oral SCC were imaged with whole body 18FDG‐PET/CT to determine its value as a diagnostic imaging and staging tool and fine needle aspirates were obtained of accessible regional lymph nodes. All tumors were FDG avid and conspicuous on 18FDG‐PET/CT images, with an average of the maximum standardized uptake value 9.88 ± 5.33 SD (range 2.9–24.9). Soft tissue infiltrative tumors that were subtle and ill defined on CT were highly visible and more extensive on FDG‐PET/CT. Tumors invading the osseous structures were more similar in extent on 18FDG‐PET/CT and CT although they were more conspicuous on PET images. Three cytologically confirmed metastases were hypermetabolic on PET, while two of those metastases were equivocal on CT.     

The use of magnetic resonance imaging for the assessment of distal limb wounds in horses: A pilot study

The utility of magnetic resonance (MR) imaging in the evaluation of equine solar foot penetrations is well established. The objective of this pilot study was to evaluate the utility of MR imaging in assessment of equine distal limb wounds excluding solar penetrations. Low-field MR images of 23 horses that had previously sustained distal limb wounds were reviewed in consensus by two ECVDI diplomats. Structures (bone; synovial structure; subcutaneous tissue and skin; and ligament/tendon) were identified as normal or abnormal on MR images, radiographs and ultrasound images and reports. All abnormalities were described. The presence of artefacts and their effect on image interpretation were also noted for each modality. Comparisons were made between imaging modalities, and it was noted if MR imaging influenced case management. Abnormalities of the bone were identified in 26% of horses on MR images and 17% of horses on radiographs; there were no osseous abnormalities identified on radiographs that were not identified on MR images, and additional features and better characterisation of lesions were noted on MR images. Tendon/ligament abnormalities were identified in 57% horses on MR and 47% of horses on ultrasound images. Magnetic susceptibility artefacts compromised MR image interpretation in 17% of horses. MR imaging of equine distal limb wounds allowed identification of both osseous and tendon/ligament abnormalities in more cases than either radiography or ultrasonography, and altered case management in 20/23 horses. Although MR imaging should not replace conventional imaging, this study highlights that MR imaging of equine distal limb wounds can yield information not detected on conventional imaging which may direct treatment and affect prognostication.     

Bioavailability Estimation of DL-Methionine Hydroxy Analogue Relative to DL-Methionine in Layers Fed Milo-Soybean Diets Using Different Regression Models

The bioavailability of liquid DL-methionine hydroxy analogue-free acid (MHA-FA) relative to dry DL-methionine (DLM) in laying hens was estimated using milo-soybean diets, which were formulated to have low methionine and cystine content to increase the sensitivity of methionine deficiency. Five graded supplemental levels of methionine (0.02, 0.04, 0.06, 0.08, and 0.10%) from DLM or MHA-FA were added to the basal diet containing 0.20% methionine on an equimolar basis. Five different models were used to determine the bioavailability of MHA-FA relative to DLM. Three of the models were exponential models with supplemental methionine concentration on a weight basis, supplemental methionine intake on a weight basis, and supplemental methionine intake on a molar basis as the independent variable, respectively. Two of the models were sloperatio models with supplemental methionine intake on a weight basis or supplemental methionine intake above basal diet on a weight basis as the independent variable. The results of the regression analysis indicated average bioavailabilities were 122 (egg production), 109 (egg mass), and 89% (egg weight) on a weight basis or 139 (egg production), 124 (egg mass), and 101% (egg weight) on a molar basis. All bioavailability values were not different (P > 0.05) from 88% on weight basis or 100% on a molar basis but were different (P < 0.05) from 65% on weight basis or 74% on a molar basis.     

Effect of tulathromycin on the carrier status of Actinobacillus pleuropneumoniae serotype 2 in the tonsils of pigs

The effect of a single or double dose of tulathromycin was evaluated in pigs carrying Actinobacillus pleuropneumoniae serotype 2 in their tonsils. Twenty-nine pigs from a reinfected specific pathogen-free-herd were selected from animals testing positive in an A pleuropneumoniae serotype 2-specific pcr test on tonsil scrapings and they were divided into three groups. The pigs in group 1 were treated subcutaneously with 2.5 mg/kg tulathromycin on day 0, the pigs in group 2 were treated with 2.5 mg/kg tulathromycin on days 0 and 4, and the pigs in group 3 were left untreated as controls. The pigs were tested by pcr on tonsil scrapings on days 0, 4, 11 and 33, and on day 33 all the animals were euthanased. There were no significant differences between the numbers of PCR-positive animals in the three groups on any of the sampling dates.     

鸡微卫星DNA标记与肉品质性状关系研究

利用25个微卫星标记，对以隐性白羽鸡和仙居鸡为亲本建立的资源群体F2500只鸡进行遗传检测，并测定各个体的鸡肉pH值、失水率、嫩度和肌间脂肪含量，采用方差分析法对标记与性状进行相关分析。结果表明：各位点平均等位基因数为5，平均杂合度为0．7012，平均多态信息含量为0．6468。嫩度间与pH值存在显著的正相关（P〈0．05），与肌间脂肪存在极显著的正相关（P〈0．01），与失水率之间存在显著负相关（P〈0．05）。方差分析显示：ADL136对肌间脂肪含量的影响极显著（P〈0．01）；MCW0095和ADL166对嫩度影响显著（P〈0．05）；MCW264对pH值影响显著（P〈0．05）；MCW264、ADL211和MCW0223对失水率影响极显著（P〈0．01）；其他标记对性状的影响未达到显著水平。