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91.
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We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns
obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods,
i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction
followed by DNA extraction, and combined RNA/DNA extraction from the bacterial cell fraction, were performed. Crude extracts
were purified and amplified using universal bacterial primers. PCR products were then analysed by DGGE, and similarity between
the profiles obtained was determined by unweighted pair group with mathematical averages clustering. The results showed clear
profiles that presumably represented the dominant bacterial fractions in the samples. The profiles generated by all four methods
were similar, indicating that the methods were of approximately equal efficiency in the extraction of target DNA representative
of the soil bacterial community. However, the patterns of clustering also indicated that different populations of bacteria
could be detected in the same soil using different soil DNA extraction methods. The application of two dilution levels of
DNA in PCR-DGGE showed that the most stable profile of the soil bacterial community could be generated by the direct methods.
The indirect methods gave clustered profiles at both dilution levels. It is likely that these methods extracted DNA from a
major, easily desorbed, bacterial fraction, consisting of low-density populations. PCR-DGGE was found to be a suitable technique
with which to assess differences in methods for DNA extraction from soil, which can be further used for the determination
of microbial community diversity at the molecular level.
Received: 22 June 1999 相似文献
94.
Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens 总被引:1,自引:0,他引:1
Introduction1Genetictransformationinconifershasthepotentialtoallowtheselectiveimprovementofindividualtraitsinelitecloneswhilestillmaintainingtheexistingcombinationofgenesresponsibleforthesuperiorphenotype(Charestetal.1991;Jamesetal.1996;Walteretal.1999).Atpresent,althoughconsiderableresearchefforthasbeendevotedtothegeneticengineeringofconiferspecies(Sederoffetal.1986;Bekkauoietal.1988,1990;Robertsonetal.1992;Bomminenietal.1993;Shinetal.1994;Klimaszewskaetal.1997),ithaslaggedbehindadvancesma… 相似文献
95.
AIM and METHODS: The ratio of mitochondrial DNA (mtDNA) deletion was measured to find the relationship between mtDNA deletion and aged learning and memory deficit. The aged rats were divided into two groups, aged learning and memory deficit group and aged learning and memory normal group. The ratio of mtDNA deletion was measured by dilution polymerase chain reaction. RESULTS: There are deleted mtDNA (about 4834 bp) in the cerebral cortex, hippocampus and cerebellum of both young and aged rats. The ratios of deleted mtDNA were similar in the cerebral cortex,hippocampus and cerebellum of young rats (about 0.00018%). The ratio mtDNA of aged learning and memory normal rats had increased by five-fold in the cerebral cortex and hippocampus, or one-fold in the cerebellum over young rats. The ratio of aged learning and memory dificit rats had increased by one-fold in the cerebral cortex or 0.8-fold in the hippocampus or two-fold in the cerebellum over aged learning and memory normal rats.CONCLUSIONS: There was really the increase of mtDNA in aging rat brain. And this increase was double in amount in aged learning and memory deficit rats compared to the normal learning and memory aged rats. It is suggested that the mtDNA deletions in the brain regions associated with learning and memory may be contributed to the cellular and molecular mechanism of learning and memory deicit with aged rats. 相似文献
96.
聚合酶链反应检测猪细小病毒的研究 总被引:17,自引:1,他引:17
本研究成功地建立了检测猪细小病毒(PPV)的聚合酶链反应(PCR)技术。以16个核苷酸引物对首次完成了PPVDNA结构蛋白VP1编码区228bp片段的扩增。同样数目的另一引物对,也成功扩增了Vp2编码区158bpDNA片段。PPVBM-1株与其它国内分离株(广西株、天津株和哈尔滨株)均出现相同扩增结果。琼脂糖凝胶电泳显示了特异条带(VP1228bp.Vp2158bp),而伪狂犬病病毒、犬细小病毒均未扩增,表明了本方法的特异性。同时,限制性内切酶分析(Vp1228bp及Vp2158bp分别含单一PvuⅡ、EcoRⅠ位点),也证实了特异性。本方法敏感性高,可检出10fg模板DNA。既可作样品的快速检测,又能检查细胞系的污染。具有特异、简便、快速的优点。 相似文献
97.
98.
应用多重PCR检测人工感染鸡呼吸道疾病的研究 总被引:3,自引:0,他引:3
本文报道了IBV,NDV,ILTV,MG人工感染4周龄SPF鸡和非免疫鸡后,用多重PCR检测试验鸡的咽喉棉拭子和器官组织样品,并与传统的病原分离鉴定,血清学方法进行比较,多重PCR无论是对单一感染病原,还是对两种以上混合感染病原,其敏感性和检测速度都优于传统的鉴别诊断方法,具有较高的实用价值,可以直接应用于临床检测,服务于生产。 相似文献
99.
鸡新城疫-传染性支气管炎-减蛋综合征三联油乳剂苗的研制及应用 总被引:1,自引:0,他引:1
用新城疫病毒(克隆H88株。)、传染性支气管炎病毒(JP株和H52株)和减蛋综合征病毒(H91株)分别接种于鸡胚和鸭胚,并收取其含毒鸡、鸭胚尿囊液,用一定浓度甲醛溶液灭活,按一定比例混匀,以7号白油为佐剂制成鸡新城疫(ND)-传染性支气管炎(IB)-减蛋综合征(EDS76)三联油乳剂灭活苗(三联苗)。三联苗免疫30日龄雏鸡,免疫后7天产生免疫应答,免疫后21天保护率达100%。疫苗在4℃~8℃保存期为12个月,10℃~25℃保存期为6月,该疫苗在全省不同地区鸡群应用400余万羽份,取得满意效果。 相似文献
100.
鲤疱疹病毒2型和3型主要感染鲫(金)鱼(Carassius auratus)、鲤鱼(Cyprinus carpio)和锦鲤(Cyprinus carpio haematopterus)及其杂交种,具有高度的传染性和致病性,对养殖鲤科鱼类危害严重。为了建立快捷、高效且能同时检测这2种类型鲤疱疹病毒的检测技术,本研究根据鲤疱疹病毒的DNA聚合酶基因(2、3型)、解旋酶基因(2型)和胸苷激酶基因(3型)的保守序列,设计了3对特异性引物,通过对三重PCR的反应条件进行优化,建立起鲤疱疹病毒2型和3型三重PCR检测方法,并运用该方法对实验室保存的鲫鱼和鲤鱼组织样品进行检测。结果显示,该三重PCR检测方法仅在鲤疱疹病毒阳性样品中扩增出3条特异性条带,表现出良好的特异性;以克隆的目的基因质粒为模板,进行10倍梯度稀释,该检测方法能检出的极限值为2.85×102 copies/μL,表现出较高的灵敏性;运用该方法检测122份鲫鱼和60份鲤鱼样品,其结果与运用国家标准(GB/T 36194-2018)和行业标准(SC/T 7212.1-2011)方法检测的结果一致。本研究表明,构建的三重PCR检测方法不仅具有较高的准确性和灵敏度,还能同时检测2种类型的鲤疱疹病毒,有效提高检测效率。 相似文献