穿山甲标本和甲片的DNA提取及PCR扩增

为验证经处理后的穿山甲(Manis spp.)标本和甲片是否可以用于种间分子鉴定标记的开发及个体识别工作,本文在样品的预处理、消化、提取后纯化等方面对传统提取方法进行了改进,分别从穿山甲剥制标本、干皮标本及甲片中提取总DNA;然后用Cyt b基因扩增通用引物、12S rRNA基因全序列扩增引物、RAPD引物及微卫星引物进行了PCR扩增,并对部分扩增结果进行了序列测定.结果表明,除剥制标本的脚底皮张组织外,其他样品基本都可以提取出DNA.以此为模板的PCR扩增中,2种线粒体基因引物扩增出明显目的条带,RAPD引物扩增出种间特异条带,测序结果可用于种间特异性引物及SCAR引物的开发;微卫星引物在甲片样品中扩增稳定,可用于个体识别工作.     

伪品乌梢蛇PCR鉴别和蛇种溯源调查

目的利用基因序列同源性分析的方法对伪品乌梢蛇蛇种溯源调查。方法 (1)用PCR的方法对供试品进行鉴别;(2)利用通用引物扩增乌梢蛇对照药材和供试品COⅠ基因,将PCR产物进行测序,测序结果在NCBI进行BLAST序列同源性比对分析。结果 (1)在与对照药材凝胶电泳图谱300～400 bp处,无DNA条带,200～300 bp处有DNA条带;(2)基因序列同源性比对结果显示,对照药材为乌梢蛇,两个供试品均为灰鼠蛇。结论用PCR方法鉴别乌梢蛇是一种准确、客观的方法。利用COⅠ基因通用引物扩增的PCR产物进行测序和序列同源性比对,可以对蛇种进行溯源调查。     

人参细胞器核糖体小亚单位DNA的PCR扩增与序列测定

采用CTAB法提取了人参(Panax ginseng)根基因组DNA,根据植物叶绿体16S rDNA和线粒体18SrDNA与细菌16S rDNA序列具有高度同源性,用扩增细菌16S rDNA的一对通用引物(8f,1492r)扩增了人参细胞器核糖体小亚单位DNA.对扩增产物进行了克隆与测序,经多序列比对,扩增片段分别与已知植物叶绿体16S rDNA和线粒体18S rDNA具高度同源性,表明该对引物可以用来扩增绝大多数植物细胞器核糖体小亚单位DNA,可以作为鉴定植物叶绿体16S rDNA和线粒体18S rDNA的一种基本实验技术.     

真菌通用引物Its1和Its4在丝状真菌鉴定中的价值评价

目的 对真菌通用引物Its1和Its4在丝状真菌鉴定中的价值进行评价.方法 收集中山大学附属第一医院2012年1月至2012年8月间分离的丝状真菌11株,使用真菌通用引物Its1和Its4采用PCR法扩增核糖体基因,对PCR产物进行序列测定,将测序结果与GenBank中已知标准或临床菌株DNA序列比对,确定丝状真菌的菌种;同时与传统形态学鉴定方法进行比较,从而对真菌通用引物Its1和Its4在丝状真菌鉴定中的价值进行评价.结果 从DNA提取到序列测定结束,在2个工作日内即可完成.所有菌株均测序成功,测序结果与GenBank中的序列比对,烟曲霉、杂色曲霉、橘青霉、溜曲霉可以鉴定到种;黄曲霉和米曲霉、阿姆斯特丹散囊菌和冠突散囊菌由于高度同源性无法区分鉴定到种.结论 利用真菌通用引物Its1和Its4结合PCR技术和测序技术,可快速、准确将大部分丝状真菌鉴定到种.     

转化医学真菌学实例——直接提取DNA鉴定菌种及体外药敏试验指导诊治面部难辨认癣

目的报道1例由万博节皮菌(趾间毛癣菌有性期)所致面部难辨认癣,取皮损鳞屑直接提取真菌DNA做菌种鉴定,并与真菌培养鉴定结果比较,对培养的菌落直接用抗真菌乳膏做药敏实验指导治疗。方法病变部鳞屑经KOH涂片真菌镜检阳性后,取鳞屑直接提取DNA,以真菌通用引物ITS1/4做PCR扩增后测序;同时从培养生长的菌提取DNA做分子生物学鉴定;并将抗真菌乳膏加入含菌平板孔中观察抑菌圈大小。结果鳞屑直接提取的DNA与培养获得菌落提取的DNA经PCR-测序均鉴定为万博节皮菌,诊断万博节皮菌感染所致面部难辨认癣。镜检阳性后即给予特比萘芬口服(250mg/d)及1%萘替芬-0.25%酮康唑乳膏外用,每周复诊并取鳞屑镜检和培养,基于药物抑菌实验结果指导治疗5周,至临床治愈和真菌培养转阴。结论鳞屑直接提取DNA做PCR-测序能及早明确菌种,待培养菌落长出后做PCR-测序验证,直接用成品抗真菌乳膏做体外药敏实验指导临床选药,动态培养鳞屑以确定疗程。此个体化诊治方案为从临床到实验室、从实验室到临床转化医学真菌学的成功实例。     

16S rRNA PCR鉴定脆弱类杆菌

目的:应用16SrRNA序列设计PCR引物鉴别脆弱类杆菌。方法:通过脆弱类杆菌16SrRNA序列特异性位点设计引物,对4株脆弱类杆菌及大肠杆菌、乳酸杆菌、嗜热链球菌等进行PCR扩增。应用琼脂糖电泳法对PCR扩增产物进行特异性检测。结果:脆弱类杆菌在176bp左右出现特异性条带,而其他细菌均未出现特异性条带。结论:通过16SrRNA序列中特异位点设计引物进行PCR,可特异性鉴定脆弱类杆菌。     

应用PCR方法鉴别猪肉和牛肉

参考已有文献建立了一种快速检测猪肉和牛肉的PCR实验方法。首先,提取基因组DNA,设计针对目的基因的特异性引物,其次,通过PCR方法扩增特异性DNA片段,再利用琼脂糖凝胶电泳予以分离和鉴定,根据DNA分子量的大小及测序结果,鉴别、确认不同的肉类样品。本实验方法简便、特异性高,不仅可作为教学实验,也可用于肉制品的检测。     

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。     

基于rbcL-a和ITS的枸杞鉴别、遗传关系分析及ITS假基因的发现

利用rbcL-a及ITS序列作为分子标记,对14个枸杞种或品种及1个伪品白刺进行了真伪鉴别及遗传关系分析。CTAB法提取总DNA,用通用引物对rbcL-a和ITS序列扩增、克隆、测序及分析,T克隆解决部分样品ITS无法成功测序问题。rbcL-a和ITS序列通用引物可在所有样品中成功扩增,rbcL-a序列全长643 bp,在14个枸杞种或品种中完全一致,与白刺具有45bp(7%)的碱基变异。ITS序列在14个枸杞种或品种中长度变异范围为513-692 bp。采用UPGMA法构建系统树,可区分白刺与枸杞,黑果枸杞和宁夏枸杞各品种分别聚为两支,云南枸杞、韩国枸杞及黄果枸杞则聚在宁夏枸杞大分支内。rbcL-a序列可有效进行枸杞真伪鉴别,ITS序列可用于枸杞品种鉴定及遗传关系分析。此外,本研究首次揭示了枸杞品种及个体内的ITS多态性,发现在枸杞中存在ITS假基因这一现象,为未来利用ITS序列进行枸杞系统进化研究提供一些新的参考。     

不同保存方式下蝗虫组织DNA的提取及RAPD分析

为了开展蝗虫分子系统学研究,分别对冷冻、乙醇浸泡(100%、乙醇、70％乙醇)和干制蝗虫标本用饱和NaCl法进行了基因组DNA的提取,并用随机引物进行扩增,结果表明：70％乙醇固定的标本和部分干标本提取的总DNA得率较低,在琼脂糖凝胶电泳检测中大音琏分有明显降解,导致PCR扩增中信息缺失,甚至无扩增条带;而保存完好的干标本、-20℃冷冻标本和100%,乙醇浸泡标本提取的总DNA带型整齐,无拖尾,PCR扩增结果的稳定性好,成为蝗虫分子系统学研究中首选的三种保存方式。     

一种直接用于PCR扩增的山羊瘤胃微生物DNA提取方法的建立

目的建立提取高质量的瘤胃微生物DNA的方法,为采用免培养技术研究山羊瘤胃微生物奠定基础。方法采集山羊瘤胃内容物,用SDS高盐法提取微生物总DNA,以通用引物扩增细菌和古细菌的16SrDNA。结果提取到的瘤胃微生物总DNA片段大于23kb,PCR能够扩增出细菌和古细菌的16SrDNA片段。结论用该提取方法得到的山羊瘤胃微生物总DNA能够满足后续实验的需要。     

High universality of matK primers for barcoding gymnosperms

DNA barcoding is a tool to provide rapid and accurate taxonomic identification using a standard DNA region. A two-marker combination of rnatK+rbcL was formally proposed as the core barcode for land plants by the Consortium for the Barcode of Life Plant Working Group. However, there are currently no barcoding primers for matK showing high universality in gymnosperms. We used 57 gymnosperm species representing 40 genera, 11families and four subclasses to evaluate the universality of nine candidate matK primers and one rbcL primer in this study. Primer (1F/724R) of rbcL is proposed here as a universal primer for gymnosperms due to high universality. One of the nine candidate matK primers (Gym_F1A/Gym_R1A) is proposed as the best "universal" matK primer for gynnosperms because of high polymerase chain reaction success and routine generation of high quality bidirectional sequences. A specific matK primer for Ephedra was newly designed in this study, which performed well on the sampled species. The primers proposed here for rbcL and matK can be easily and successfully amplified for most gymnosperms.     

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis.     

Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs

Background

Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.

Results

We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.

Conclusion

We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.     

Generation of a Sequence Characterized Amplified Region Probe for Authentication of Mainland Serow (Capricornis sumatraensis)

Mainland serow is an endanged artiodactyl of southern Anhui province, China, that is often subject to poaching. To provide an easy, rapid and reliable marker for identification of bushmeat, skin and other tissues of the species, we developed a sequence characterized amplified region (SCAR) based on a species-specific random amplified polymorphic DNA (RAPD) marker. Initially, a 1012-bp species-specific DNA fragment of mainland serow was detected by a RAPD primer S1193. Then, a serow-specific primer pair (SCF/SCR) was designed according to the specific RAPD fragment, resulting in a 438-bp SCAR for the species. Finally, the reliability of the SCAR primers was tested by a common multiplex polymerase chain reaction using the combination of the SCAR and cyt b universal primers. The results that all mainland serow samples presented two target bands but the others failed to produce the SCAR indicated that the designed primers were highly diagnostic. Therefore, the SCAR probe developed in this study will be useful for quick authentication of mainland serow tissue samples for conservation biology and bushmeat regulation.     

RAPD标记在山葡萄种质鉴定中的应用

采用修改后的CTAB法获得了高质量的基因组DNA.利用RAPD标记技术对山葡萄7份种质进行鉴定,用4个引物(从30个引物中筛选)对试材进行PCR扩增,共扩增出30条谱带,平均每条引物产生7.5条谱带,其中21条谱带为多态性谱带,占总谱带数的70%.不同引物扩增的谱带数不同,范围在6～9条之间.利用4个引物扩增出的多态性谱带可以将7份山葡萄种质区分.     

A Next Generation Semiconductor Based Sequencing Approach for the Identification of Meat Species in DNA Mixtures

The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.     

An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications

We proposed a modification the procedure of genotyping based in labeled universal primer and tailed primer. In the standard protocol, three primers are used in the same PCR reaction, a forward primer with tail added at the 5′ end of the identical sequence to labeled universal primer with dye-fluorescent and a reverse primer. Unfortunately, the choice of a labeled primer characterized by a large number of complementary sequences in target genomes (which is more probable in larger genomes) result in unspecific amplifications (false positive) can cause absence or decrease amplification of the locus of interest and also false interpretation of the analysis. However, identification of possible homologies between the primer chosen for labelling and the genome is rarely possible from the available DNA data bases. In our approach, cycling is interrupted for the addition of the labeled primer only during the final cycles, thus minimizing unspecific amplification and competition between primers, resulting in the more fidelity amplification of the target regions.     

DNA条形码技术在毒品原植物大麻鉴定中的应用

准确鉴定毒品原植物大麻的种属及品种具有重要的理论和实践意义。为了探讨DNA条形码技术用于毒品原植物大麻种属鉴定及品种鉴定的可行性,该研究以60份大麻原植物(分别采自内蒙、黑龙江、陕西延安、陕西榆林4个地区的栽培大麻雌雄各6株及新疆玛纳斯地区的野生大麻雌雄各6株)为材料,通过从其叶片中提取的DNA为模版,利用核糖体DNA基因间隔区的通用引物ITS2和叶绿体DNA的通用引物psbAtrnH进行PCR扩增,对扩增片段进行双向测序,将测序结果进行人工矫正和比对。结果显示:所有大麻样本的ITS2扩增片段序列没有变异完全一致,但psbA-trnH扩增片段变异较大共检测出8种cpDNA单倍型,用MEGE5.1软件计算种间遗传距离,并构建NJ系统聚类树可以有效把这五个地区的大麻样本区别开来,因此证明DNA条形码技术在毒品原植物大麻的种属鉴定方面具有可行性,但其用于大麻的种属鉴定的准确性、可靠性及在其来源地鉴定及品种鉴定中的可能性还有待进一步深入地研究。