同科植物SSR引物在烟草遗传差异分析中的应用研究

为丰富分子标记类型,加速烟草种质资源遗传多样性的研究利用,从番茄、辣椒的SSR引物中选择22对SSR引物,用遗传差异较大的14个烟草材料筛选引物,应用筛选出的引物对24份烤烟材料进行扩增,分析其遗传差异性。22对SSR引物中筛选出8对重复性好、多态性高的引物。8对引物在24份烤烟材料中共检测到98条遗传多态性带,引物多态信息量变化范围为0.451～1,平均0.646。品种间的遗传相异系数变异范围为0.3445～0.8626,平均0.6052。引物9扩增的谱带可把24个品种完全区分开。在遗传距离为0.62水平可将24份材料聚为四大类,其聚类结果与实际亲缘关系基本一致。因此,同科植物的SSR引物能较好地从分子水平上揭示烟草种质资源遗传背景和亲缘关系,可以应用于烟草品种资源的遗传差异分析。     

微生物酶法降解烟草总植物碱试验

为寻求有效降低烟草中烟碱含量的方法,利用微生物酶法进行了降解烟草烟碱试验。所用的酶为烟叶微生物经优选、超临界二氧化碳破壁后调制获得的多酶体系。烟叶被不同剂量的酶制剂喷洒后,在不同时间取样评吸并测定其烟碱含量。结果表明:①酶处理烟草烟碱的降低速度比自然陈化的提高了几十倍至几百倍;②在酶用量一定的条件下,烟碱的降低量随着作用时间的延长而增大,但至4d后烟碱的含量几乎不再变化;③在作用时间一定的条件下,烟叶中的烟碱含量与酶用量基本上呈负相关关系,即酶用量越大,烟叶中的烟碱含量越低;④酶法在降解烟草烟碱方面优于微生物法,并且具有可控制性。     

烟草品种的SCAR标记鉴别

利用6对SCAR引物对12个烟草(Nicotiana tabacum L.)品种复烤叶片的基因组DNA进行SCAR-PCR扩增分析。根据6个SCAR标记在不同烟草品种中的扩增差异,建立了12个烟草品种的分子ID和鉴别路线,分析了SCAR标记鉴别遗传差异较小烟草品种的可行性。结果表明:利用组合标记策略,6个SCAR标记可准确地区分12个烟草品种中具有相同抗感性,亲缘关系较近的烟草品种,并可用于复烤叶片的品种鉴定。     

烟草野火病菌特异性检测引物筛选及应用

烟草野火病是山东烟区主要的细菌性病害之一,为了能够对该病害进行精确、快速的分子鉴定,本研究从NCBI基因组数据库中下载了假单胞菌属25个不同菌种和127个丁香假单胞菌属不同致病变种的全基因组数据,利用Mauve 2.3.1进行全基因组比对,获得丁香假单胞菌烟草致病变种特异性区段。在此基础上利用Primer Premier 6.0进行引物设计,筛选出了一对丁香假单胞菌烟草致病变种的特异性检测引物40429。利用7株山东不同烟区的烟草野火病菌和4株分别来自于贵州、湖北、福建及云南的烟草野火病菌进行了该引物的适用性验证,结果表明该引物均可扩增得到大小为203 bp的单一目的条带。说明该引物对于烟草野火病菌的检测具有良好的适用性,可以用于野火病菌的分子鉴定。     

基于烟草属特异性基因Ntsp151的环介导等温扩增检测

针对烟草属特异性基因Ntsp151序列保守区设计环介导等温扩增(LAMP)引物,基于SYBR Green I荧光染料对烟草材料进行可视化LAMP检测,对检测过程中的DNA提取方法和反应条件进行优化,并对LAMP反应体系的特异性和灵敏度进行评价。结果表明：使用Chelex-100提取的DNA可以直接进行LAMP扩增反应,显色效果较好;LAMP最适反应条件为扩增温度63℃、反应时间60 min、Mg²⁺浓度6 mmol/L;LAMP对17份非烟草材料和1份烟草材料基因组DNA的扩增显色结果与荧光值的检测结果一致,具有较好的特异性,其最低检测限为10² copies/μL。基于烟草属特异性基因Ntsp151的LAMP检测结果可视化强,且灵敏度高、特异性强,适用于现场抽检。     

烟草甲和烟草粉螟的防治研究

简述了烟草甲（Ｌ．ｓｅｒｒｉｃｏｒｎｅ）和烟草粉螟（Ｅ．ｅｌｕｔｅｌｌａ）在烟叶仓储中的危害情况,综述了国内外储烟害虫防治技术研究进展。在此基础上,针对我国在烟叶储存养护和卷烟生产线上如何防虫杀虫的问题,提出了初步的建议。     

基于RAPD分子标记的烟草青枯病菌特异引物筛选及效果评价

为建立烟田土壤和烟株烟草青枯病菌的快速诊断方法,本研究采用RAPD方法筛选获得了6对可用于烟草青枯病菌PCR检测的特异性引物.经对6种细菌(烟草青枯病菌、多粘芽孢杆菌、枯草芽孢杆菌、节杆菌、荧光假单胞菌、野火病菌)和2个烟草品种(中烟100和云烟87)的检测验证,6对引物均具有良好的灵敏度、特异性和可靠性.对青枯菌菌液...     

烟草中游离烟碱与其pH值之间的关系

研究了烟草中游离烟碱的萃取条件 ,确定了采用水、三氯甲烷依次萃取和气相色谱分析烟草中游离烟碱含量的方法 ,烟碱的回收率为 95 .1%～ 99.8% ,方法的RSD为 1.2 1%～ 1.70 %。同时 ,还考察了烟草总烟碱及其pH值测定方法的准确性 ,采用这些方法测定了 4 7种商品卷烟的游离烟碱、总烟碱和pH值 ,并进行了相关分析。结果表明 ,游离烟碱、游离烟碱与总烟碱的比值和pH值都呈显著的正相关关系 ,游离烟碱和总烟碱、总烟碱和pH值均无相关性     

造纸法烟草薄片萃取技术研究

萃取是造纸法烟草薄片制造工艺中的关键工序之一。烟梗、烟末用水萃取,考察了萃取温度、料液比、萃取时间等对萃取率的影响。结果表明,烟梗最佳萃取工艺条件为：萃取温度60℃,料液比1：5,萃取时间70min;烟末最佳萃取工艺条件为：萃取温度50℃,料液比1：5,萃取时间50min。在最佳萃取工艺条件下,烟梗和烟末的萃取率分别为48．52％和53．76％。     

烟草中烟碱的高效液相色谱分析综述

综述了烟草及烟草制品中烟碱的高效液相色谱分析的研究现状、反相和离子对HPLC色谱模式、样品提取与辅助技术和纯化等,认为应加强有关建立快捷、稳定、对环境友好的处理大量样品的制备方法研究。     

PorA specific primers for the identification of Campylobacter species in food and clinical samples

Campylobacteriosis is a public health problem with considerable socio-economic impact. As the European Food Safety Authority has emphasized the importance of a surveillance programme for campylobacteriosis, the aim of the present study was the optimization of a specific and sensitive PCR protocol able to detect Campylobacter species responsible for gastrointestinal infections. Raw poultry meat samples were analysed for the presence of Campylobacter sp., by plating onto mCCD (Modified Charcoal-Cefoperazone-Deoxycholate) Agar and Campylobacter Selective Preston Agar and using four sets of species-specific primers for Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis and Campylobacter lari designed to bridge the porA gene. The resulting primers demonstrated a sensitivity of 0.01 ng/μl for the C. coli-specific, C. lari-specific, and C. upsaliensis-specific primer sets and 0.5 ng/μl for the C. jejuni-specific primer sets using DNA from pure cultures. Non-specific amplification of non-target DNA was not observed indicating excellent specificity. The primers were useful for the analyses of poultry meat samples both for direct plating onto mCCDA, and for DNA extracted directly from the cells grown for 48 h in Preston enrichment broth. The sets of primers were also useful when used for species identification of human isolates.     

啤酒有害乳酸菌PCR引物的设计与验证

根据乳酸菌16SrDNA序列的特征，设计合成了针对啤酒有害乳酸菌的通用引物，在16SrDNA基因水平上采用聚合酶链式反应（PCR）技术鉴定了啤酒中59株有害乳酸菌的种类。同时根据鉴定结果设计了8种乳酸菌的特异引物，PCR技术验证结果表明该8种特异引物能够准确鉴定乳酸菌。     

Specific identification of certain probiotic Lactobacillus rhamnosus strains with PCR primers based on phage-related sequences

PCR primers derived from Lactobacillus rhamnosus phage Lc-Nu genome were used to screen the presence of phage-related sequences in Lb. rhamnosus strains. Several primer pairs derived from structural and replication gene regions of phage Lc-Nu amplified PCR products of expected sizes from bacterial strains revealing phage-related sequences in 10 of 11 Lb. rhamnosus strains. Strain-specific PCR primers for three probiotic Lb. rhamnosus strains were derived from these phage-related sequences for identification and detection purposes. Specificity of these primers was tested against 11 Lb. rhamnosus strains and over 40 other bacterial strains.     

Selection of primers for specific detection of Clostridium botulinum types B and E neurotoxin genes using PCR method

Improved oligonucleotide primers were designed to flank 370- and 307-bp fragments of the bont genes encoding botulinum neurotoxins types B and E, respectively. Primer specificity was confirmed for reference strains of Clostridium botulinum types B and E for strains representing bacterial species common in food, and for the DNA mixtures of C. botulinum types B and E in the presence of background DNA isolated from cold smoked salmon and ham. The detection limit of template DNAs of C. botulinum types B and E from the DNA mixtures increased from 1 to 0.1 ng by raising annealing temperature from 50 degrees C to 62 degrees C.     

Development of novel species‐specific primers for species identification of the Lactobacillus casei group based on RAPD fingerprints

BACKGROUND: It is difficult to clearly distinguish and identify specific species of the Lactobacillus casei group using phenotypic and genotypic (16S ribosomal DNA sequence analysis) techniques alone. Some species of this group are probiotic and are widely used in the food and feed industries. The objective of this study was to develop species‐specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting for species identification within the closely related L. casei group of bacteria. RESULTS: Three random primers termed OPT‐14, OPA‐11 and OPT‐16 were developed for analysis. The primer pairs each produced a species‐specific band found only in the tested Lactobacillus rhamnosus, Lactobacillus paracasei subsp. tolerans and Lactobacillus zeae isolates respectively. These specific fragments were then sequenced for further analysis. The species‐specific primers were designed according to cloned sequencing, which was employed for polymerase chain reaction (PCR) with the template DNA of Lactobacillus strains. Single 102, 179 and 451 bp species‐specific bands were found only in L. rhamnosus, L. paracasei subsp. tolerans and L. zeae respectively. CONCLUSION: Using PCR, the novel species‐specific primers have been shown to rapidly, accurately and effectively identify species of L. rhamnosus, L. paracasei subsp. tolerans and L. zeae from within the L. casei group of probiotic bacteria. Copyright © 2009 Society of Chemical Industry     

PCR identification of Salmonella serogroups based on specific targets obtained by comparative genomics

Comparative genomic approaches provide abundant information to reveal the diversity among Salmonella serogroups. In a local genomic sequence database, twenty-five Salmonella whole genomic sequences were divided into 6 (A, B, C1, C2, D and others) serogroups for mining the DNA fragments specific for serogroups A through D. For each serogroup, a reference sequence was selected and split into 1000-bp fragments in silico to align against all the other genomic sequences to obtain one or more serogroup-specific fragments. As a result, 2, 6, 7, 10, and 7 specific fragments were found for A, B, C1, C2 and D serogroups, respectively. Specific primer sets targeting these DNA fragments were designed for multiplex PCR assays identifying 21 Salmonella standard strains and 86 additional food isolates. The PCR results demonstrated good agreement with those from Salmonella serotyping. This means that the PCR assay may be able to identify 5 (A, B, C1, C2 and D) Salmonella serogroups and elucidate differences among them. Based on the gene annotations, the 32 serogroup-specific fragments were divided into 3 categories (membrane protein genes, rfb gene clusters, and fimbrial genes). Each gene from these three groups was conserved within the serogroup and was closely correlated with phenotypic characterization. This finding implies that these genes, which are associated with sugar synthesis and metabolism or glycosyl and O-actetyl transfer, impart the differences among Salmonella serogroups.     

Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars

Salmonella enterica serovar Typhimurium is a major foodborne pathogen throughout the world. Until now, the specific target genes for the detection and identification of serovar Typhimurium have not been developed. To determine the specific probes for serovar Typhimurium, the genes of serovar Typhimurium LT2 that were expected to be unique were selected with the BLAST (Basic Local Alignment Search Tool) program within GenBank. The selected genes were compared with 11 genomic sequences of various Salmonella serovars by BLAST. Of these selected genes, 10 were expected to be specific to serovar Typhimurium and were not related to virulence factor genes of Salmonella pathogenicity island or to genes of the O and H antigens of Salmonella. Primers for the 10 selected genes were constructed, and PCRs were evaluated with various genomic DNAs of Salmonella and non-Salmonella strains for the specific identification of Salmonella serovar Typhimurium. Among all the primer sets for the 10 genes, STM4497 showed the highest degree of specificity to serovar Typhimurium. In this study, a specific primer set for Salmonella serovar Typhimurium was developed on the basis of the comparison of genomic sequences between Salmonella serovars and was validated with PCR. This method of comparative genomics to select target genes or sequences can be applied to the specific detection of microorganisms.     

基于物种特异性PCR对海鳗鱼糜掺假检测技术的研究

为了对海鳗鱼糜掺假进行定性研究,从GenBank数据库下载海鳗及其他物种的线粒体12S rRNA,并通过BioEdit 7.0软件对该基因碱基序列进行比对。在此基础上,综合考虑引物设计原则,设计出关于海鳗的特异性引物。对引物的特异性、灵敏性以及主要加工过程进行实验,结果表明所设计的引物对海鳗DNA具有很强的特异性,灵敏性达到0.1%,加工过程也不影响检测过程,从而满足了对于海鳗鱼糜掺假的检测要求。     

基于blaCARB-17和toxR基因的副溶血弧菌双重PCR检测方法的建立

建立一种针对副溶血弧菌bla_CARB-17和toxR基因的双重PCR检测方法。按照副溶血弧菌的bla_CARB-17和toxR基因序列设计并合成引物,于同一反应体系中进行PCR扩增,优化退火温度、退火时间和引物比例,确定双重PCR的特异性和灵敏性,最后用实验室分离鉴定的副溶血弧菌分离株验证。结果表明:建立的双重PCR最佳退火条件是52℃30 s,引物比例1∶1,在同一体系中特异地扩增出副溶血弧菌的bla_CARB-17和toxR基因的303、350 bp片段,其它对照菌株均无扩增,表明该方法特异性良好。双重PCR的最低检测限达1×10² CFU/mL,对实验室鉴定的56个副溶血弧菌分离株验证均为阳性。建立的双重PCR方法操作简单、高效快速、特异性强,适用于副溶血弧菌的检测。