通用引物双重PCR在节肢动物物种鉴定中的应用

【目的】本研究旨在使用基于线粒体基因通用引物的双重PCR技术同时扩增单一样本中两条标记基因,从而达到简化节肢动物物种鉴定流程的目的。【方法】在一次PCR实验中同时加入可扩增线粒体COI基因和16S rDNA两个不同分子标记的引物,对3纲8目14科的14种节肢动物物种标本的基因组DNA进行扩增;扩增产物经电泳和胶回收后测序,并BLAST在线搜索相似序列,验证基于通用引物的双重PCR在不同的动物类群中用于物种鉴定的有效性。【结果】应用基于COI和16S rDNA的引物从分属于3纲8目14科的14种节肢动物基因组DNA中均可成功扩增目的基因;扩增产物测序结果进一步证实了扩增的准确性。【结论】通过本方法进行物种的分子鉴定,不仅可以保证物种鉴定的高准确率,还可以明显减少时间与DNA样本量的消耗,这对需要快速准确鉴定物种或珍稀的材料样本十分重要。     

DNA条形码试剂盒检测技术在大小蠹属种类鉴定中的应用（英文）

【目的】DNA条形码技术已成为生物分类鉴定的有力工具。DNA条形码技术的相关问题,如物种种内和种间的遗传距离出现重叠区域,将直接影响到物种鉴定的准确性。我们应用DNA条形码试剂盒检测技术来快速、准确地鉴定口岸截获的检疫性大小蠹属种类。【方法】针对大小蠹昆虫设计引物以提高PCR扩增效率。运用自主研发的基因条码分析软件找出基因片段上区分每个物种的多态位点规律,作为该物种的鉴定特征并建立数据库,应用于物种鉴定。【结果】使用针对大小蠹属昆虫设计的引物成功扩增出325 bp的COI基因片段。将大小蠹属12种昆虫的COI基因片段上的核苷酸诊断位点的组合作为物种的鉴定特征,可以准确地区分近似种。通过比对植物检疫鉴定系统数据库里的鉴定特征,将6个大小蠹属的未知样品成功鉴定到种(核苷酸序列一致性为100%),与形态鉴定结果一致。【结论】结果表明DNA条形码试剂盒检测技术可以准确鉴定大小蠹属的种类。该检测技术可以应用于其他经济重要性有害生物的检测鉴定。     

DNA条形码试剂盒检测技术在大小蠹属种类鉴定中的应用

[目的]DNA条形码技术已成为生物分类鉴定的有力工具.DNA条形码技术的相关问题,如物种种内和种间的遗传距离出现重叠区域,将直接影响到物种鉴定的准确性.我们应用DNA条形码试剂盒检测技术来快速、准确地鉴定口岸截获的检疫性大小蠹属种类.[方法]针对大小蠹昆虫设计引物以提高PCR扩增效率.运用自主研发的基因条码分析软件找出基因片段上区分每个物种的多态位点规律,作为该物种的鉴定特征并建立数据库,应用于物种鉴定.[结果]使用针对大小蠹属昆虫设计的引物成功扩增出325 bp的COI基因片段.将大小蠹属12种昆虫的COI基因片段上的核苷酸诊断位点的组合作为物种的鉴定特征,可以准确地区分近似种.通过比对植物检疫鉴定系统数据库里的鉴定特征,将6个大小蠹属的未知样品成功鉴定到种(核苷酸序列一致性为100％),与形态鉴定结果一致.[结论]结果表明DNA条形码试剂盒检测技术可以准确鉴定大小蠹属的种类.该检测技术可以应用于其他经济重要性有害生物的检测鉴定.     

PCR 芯片及其应用研究进展

PCR芯片实质上就是固定有与研究对象有关的许多已知基因的引物阵列并可用于PCR检测的固相载体,其制作时最关键的是目的基因的引物设计。与基于杂交的芯片技术不同,PCR芯片技术是一种高通量的,准确、灵敏的定量检测基因表达的技术,它将待测基因的引物固定于固相载体上,通过简单的、经过优化的定量PCR体系和荧光定量PCR仪,实现待检样品中已知基因的扩增,用于定量检测待检样品中已知基因的表达情况。PCR芯片由于其操作简单、结果准确、数据产出快而多等特点,已应用于疾病发病机制、药物作用机理和细菌分型等研究领域,并将在生命科学研究领域得到更为广泛的应用。     

mRNA差异显示条件的优化

运用优化的mRNA差异显示技术分离受内生真菌诱导的差异基因。优化差异显示条件表现在增如指定引物和随机引物的长度、改变PCR参数和再扩增程序、运用银染显色等。应用这些条件共获得7个阳性差异片段。用未优化的PCR程序1筛选35条差异带,得到3个两端均为随机引物的差示片段。而用优化的PCR程序2,52条差异带中得到9条只能用锚定引物和随机引物才能扩增出的片段。地高辛标记的反向-Northern鉴定为阳性后进行克隆和测序。PCR方法1所得的3个差示片段均无开放的阅读框。PCR程序2得到7个差异表达的基因中,2个为已知基因,5个为未知基因。因此可运用优化的差显技术分离差异表达的基因。     

DNA技术在海洋食品物种鉴定中的应用

随着分子生物学技术的发展, 海洋食品物种鉴定方法由原来的蛋白质水平深入到了DNA水平。目前应用于海洋食品物种鉴定的DNA技术主要是FINS(Forensically informative nucleotide sequencing)、PCR-RFLP和物种特异性PCR标记技术等, 能够实现对新鲜、冰冻、腌制或灌装食品物种进行鉴定, 而对混合样本的鉴定及量化分析是尚待解决的一个问题。基因数据库对物种鉴定的影响也越来越大, 是海洋加工食品物种鉴定可利用的另一种重要信息资源。文章综述了DNA技术在海洋食品物种鉴定中的应用研究进展, 并展望DNA技术在海洋食品检测中的发展趋势。     

一种新的DNA分子标记技术——起始密码子-微卫星扩增多态性

综合SCoT和ISSR分子标记技术开发了一种既能将标记位点与表达序列紧密联系,又具有相对较高的多态性的新的分子标记技术——起始密码子一微卫星扩增多态性（start codon-simple sequence repeat, SC-SSR）。SC—SSR标记是基于PCR的目的基因标记系统．上游引物用SCoT标记引物,瞄准基因区域,下游引物用ISSR标记引物,上下游引物间可自由组配。引物设计原则同SCoT标记和ISSR标记。使用50℃的退火温度,保证了扩增结果的稳定性。PCR结果采用琼脂糖凝胶电冰和聚丙烯酰胺凝胶电泳检测。SC—SSR分子标记结合了ISSR标记和SCoT标记的优点,具有操作简单、成本低廉、多态性丰富、重复性好、引物设计简单且通用性良好、同时与表达序列紧密连锁等诸多优点,可用于种质资源的鉴定评价、遗传图谱的构建、重要性状基因标记、gDNA与cDNA指纹分析乃至图位克隆等方面。     

运用多种策略改良差异显示PCR

目的：改进差异显示PCR技术,提高其在筛选差异表达基因方面的效率。方法：①采用单碱基金铆钉引物;②增加引物长度;③提高PCR反应的严谨性;④应用同步重复测序电泳。结果：减少经典方法的工作量,降低了非特异性和假阳性率。用改良技术研究热适应大鼠下丘脑基因的差异表达,发现了一个差异表达基因片段,斑点杂交证实为阳性片段。结论：改进后的差异显示PCR技术是一种研究基因差异表达从而发现新基因或基因新功能的有效方法。     

特异性检测胰腺组织表达Cre重组酶转基因小鼠的方法

利用组织特异性分子标志物启动子调控Cre重组酶,研制了6种在不同组织中特异性表达Cre重组酶的转基因小鼠.这些转基因小鼠的基因型鉴定均使用设计在Cre基因编码区的通用引物.为了特异性检测胰腺组织表达Cre重组酶的转基因小鼠,在大鼠胰岛素RIP启动子上和Cre基因上设计1对引物进行PCR扩增,并通过凝胶电泳进行分析.PCR结果显示,设计在Cre基因上的通用引物可以从6种不同组织特异性Cre重组酶转基因小鼠基因组DNA中扩增获得480 bp产物;利用本研究设计的特异性引物可以从胰腺组织表达Cre重组酶转基因小鼠基因组DNA中扩增200 bp的目的条带.这一结果表明,利用特异性引物进行PCR反应,可有效地将胰腺组织表达Cre重组酶转基因小鼠与其他多种组织的Cm重组酶转基因小鼠鉴别开来.     

格特隐球菌交配型的多重PCR鉴定

【目的】建立一种基于格特隐球菌α交配型位点内SXI1α基因和a交配型位点内SXI2a基因的多重PCR分析,用于快速鉴定格特隐球菌的交配型。【方法】设计针对格特隐球菌α交配型位点内SXI1α基因部分片段和格特隐球菌a交配型位点内SXI2a基因部分片段的特异性引物,用于多重PCR鉴定格特隐球菌的交配型;并与交配试验以及已报道的扩增α交配型位点的引物MFα、STE12α,及扩增a交配型位点的引物STE20a、STE3a进行扩增效果的比较。【结果】基于SXI1α基因和SXI2a基因的多重PCR分析,准确鉴定所有受试格特隐球菌(包括VGI、VGII、VGIII和VGIV基因型)的交配型,引物STE12α、STE20a和STE3a在常规PCR鉴定中不能鉴定部分菌株的交配型;66.7%的受试菌株不能发生交配,交配试验无法鉴定其交配型。【结论】建立的多重PCR方法明显优于常规PCR或交配试验鉴定。     

Identification of Aerobically and Anaerobically Induced Genes in Enterococcus faecalis by Random Arbitrarily Primed PCR

Enterococci have emerged among the leading causes of nosocomial infection. With the goal of analyzing enterococcal genes differentially expressed in environments related to commensal or environmental colonization and infection sites, we adapted and optimized a method more commonly used in the study of eukaryotic gene expression, random arbitrarily primed PCR (RAP-PCR). The RAP-PCR method was systematically optimized, allowing the technique to be used in a highly reproducible manner with gram-positive bacterial RNA. In the present study, aerobiosis was chosen as a variable for the induction of changes in gene expression by Enterococcus faecalis. Aerobically and anaerobically induced genes were detected and identified to the sequence level, and differential gene expression was confirmed by quantitative, specifically primed RT-PCR. Differentially expressed genes included several sharing identity with those of other organisms related to oxygen metabolism, as well as hypothetical genes lacking identity to known genes.     

Identification of aerobically and anaerobically induced genes in Enterococcus faecalis by random arbitrarily primed PCR

Enterococci have emerged among the leading causes of nosocomial infection. With the goal of analyzing enterococcal genes differentially expressed in environments related to commensal or environmental colonization and infection sites, we adapted and optimized a method more commonly used in the study of eukaryotic gene expression, random arbitrarily primed PCR (RAP-PCR). The RAP-PCR method was systematically optimized, allowing the technique to be used in a highly reproducible manner with gram-positive bacterial RNA. In the present study, aerobiosis was chosen as a variable for the induction of changes in gene expression by Enterococcus faecalis. Aerobically and anaerobically induced genes were detected and identified to the sequence level, and differential gene expression was confirmed by quantitative, specifically primed RT-PCR. Differentially expressed genes included several sharing identity with those of other organisms related to oxygen metabolism, as well as hypothetical genes lacking identity to known genes.     

Application of polymerase chain reaction with specific and arbitrary primers to identification and differentiation of Leishmania parasites

Abstract Two oligonucleotide primers Lsmc1 and Lsmv1 derived from the conserved and the variable region of a major class kinetoplast DNA (kDNA) minicircle (pLURkE3) of Leishmania strain UR6 were used for the polymerase chain reaction (PCR) in order to amplify a 461-bp fragment from the kDNAs of different Leishmania species. These primers amplify the specific fragment from the kDNAs of cutaneous species only. The cutaneous species can further be distinguished by randomly amplified polymorphic DNA (RAPD) analysis of the kDNAs of these organisms using arbitrarily chosen oligonucleotides. The arbitrary primers also generate polymorphic DNA fingerprints at the genomic level with different L. donovani isolates. The results indicate that the PCR and arbitrarily primed PCR (AP-PCR) may be extremely useful approaches for identifying and distinguishing Leishmania parasites.     

Rapid differentiation of phenotypically and genotypically similar <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> strains by PCR fingerprinting.

PCR amplification techniques viz., repetitive DNA element PCR (REP-PCR), short tandemly repeated repetitive PCR (STRR-PCR) and arbitrarily primed PCR (RAPD-PCR) were used for the taxonomic discrimination among the strains of the unicellular cyanobacterium Synechococcus elongatus collected across the coastal regions of the Indian subcontinent. These strains showed similar phenotypic and genotypic characteristics. Data obtained from genomic fingerprinting were used to perform cluster analysis and demonstrated ability to differentiate strains at intra-specific level. Polymorphisms of different PCR amplification products can serve as strain-specific molecular fingerprints. In comparison with the STRR and RAPD, the REP primer set generates fingerprints of lower complexity, but still the phenogram clearly differentiated the strains. In conclusion, described PCR fingerprinting methods can be considered as promising tools for the differentiation at the strain level of cyanobacteria from the same species.     

Isolation and expression of a gene (CGR1) regulated during the yeast-hyphal transition in Candida albicans

We used RNA fingerprinting of arbitrarily primed PCR to isolate genes upregulated during the yeast-hyphal transition in Candida albicans. The sequence and expression of one of these genes (CGR1, Candida growth regulation) are presented. Our results suggest that CGR1 expression is associated with a growth cessation of yeast cells, a prerequisite for germination in this organism.     

Screening for ribosomal-based false positives following prokaryotic mRNA differential display

Differential display (DD) and the closely related RNA arbitrarily primed PCR (RAP-PCR) have become the molecular tools of choice for identifying and isolating differentially expressed genes in both eukaryotic and prokaryotic systems. However, one of the current drawbacks of both techniques is the high number of false positives generated. In prokaryotic applications, the many false positive typically generated by DD are subsequently identified as rRNAs because of their greater abundance compared to mRNAs. To circumvent this problem, full-length 16S and 23S rDNA probes, derived from Pseudomonas putida G7 and Pseudomonas aeruginosa FRD1, respectively, were used as a prescreening approach to discriminate between those bands, which appear to be differentially expressed mRNAs, but in fact are rRNAs, following prokaryotic mRNA DD.     

Evidence for chimeric sequences formed during random arbitrarily primed PCR

Chimeric sequences were observed to occur abundantly (48% of clones) during random arbitrarily primed polymerase chain reaction (RAP-PCR) experiments designed to examine differential expression of genes involved in metal resistance in sulfate-reducing bacteria (SRB). Some of the chimeric sequences were composed of sequence from a gene differentially expressed under the imposed conditions and a sequence of the 16S or 23S rRNA gene. The remainder were composed of two rRNA sequences. Experiments using PCR and genomic sequence analysis showed that the chimeric sequences were not due to a genetic mutation (e.g., recombination, transposition). As RAP-PCR has been widely used to identify differentially expressed genes, this observation may aid in our interpretation of RAP-PCR data.     

Rapid identification of Acremonium lolii and Acremonium coenophialum endophytes through arbitrarily primed PCR

Abstract Using a random decamer 5'-CCGAGGTGAC-3' in an arbitrarily primed PCR, similar band patterns were observed between Acremonium lolii and A. coenophialum DNA, which were somewhat different from those formed by other fungal DNA. Despite sharing bands of around 0.7, 0.9 and 2.1 kb, A. lolii can be distinguished from A. coenophialum by the presence of an additional band at around 0.5 kb in the arbitrarily primed PCR.