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。     

Novel universal primers establish identity of an enormous number of animal species for forensic application

This study describes a polymerase chain reaction (PCR)‐based approach, which without knowing the history of a forensic sample, is able to reveal whether the source of the sample is human or animal, and, if animal, which of the 221 animal species included in the study, simply by using one set of novel primers to amplify and sequence the PCR amplicons. The primers described in this study universally amplify a specific segment of mitochondrial cytochrome b sequence from a sample of unknown origin and delineate its identity to the level of family, genus and species. Because these primers are universal, this approach can be applied to an enormous number of other species, which are not included in the study, and could be an ultimate solution for the identification of species for forensic application.     

Universal primer cocktails for fish DNA barcoding

Reliable recovery of the 5′ region of the cytochrome c oxidase 1 (COI) gene is critical for the ongoing effort to gather DNA barcodes for all fish species. In this study, we develop and test primer cocktails with a view towards increasing the efficiency of barcode recovery. Specifically, we evaluate the success of polymerase chain reaction amplification and the quality of resultant sequences using three primer cocktails on DNA extracts from representatives of 94 fish families. Our results show that M13‐tailed primer cocktails are more effective than conventional degenerate primers, allowing barcode work on taxonomically diverse samples to be carried out in a high‐throughput fashion.     

DNA barcoding for the identification of smoked fish products

DNA barcoding was applied to the identification of smoked products from fish in 10 families in four orders and allowed identification to the species level, even among closely related species in the same genus. Barcoding is likely to become a standard tool for identification of fish specimens and products.     

Molecular identification of cryptic bumblebee species from degraded samples using PCR–RFLP approach

The worldwide decline and local extinctions of bumblebees have raised a need for fast and accurate tools for species identification. Morphological characters are often not sufficient, and molecular methods have been increasingly used for reliable identification of bumblebee species. Molecular methods often require high‐quality DNA which makes them less suitable for analysis of low‐quality or older samples. We modified the PCR–RFLP protocol for an efficient and cost‐effective identification of four bumblebee species in the subgenus Bombus s. str. (B. lucorum, B. terrestris, B. magnus and B. cryptarum). We used a short partial mitochondrial COI fragment (446 bp) and three diagnostic restriction enzymes (Hinf I, Hinc II and Hae III) to identify species from degraded DNA material. This approach allowed us to efficiently determine the correct species from all degraded DNA samples, while only a subset of samples 64.6% (31 of 48) resulted in successful amplification of a longer COI fragment (1064 bp) using the previously described method. This protocol can be applied for conservation and management of bumblebees within this subgenus and is especially useful for fast species identification from degraded samples.     

基于种特异性COI引物（SS-COI）的桉树枝瘿姬小蜂A、B隐种快速鉴定技术

隐种A和隐种B是桉树枝瘿姬小蜂Leptocybe invasa两种重要的全球入侵隐种,对多国林业生产造成了严重危害。由于桉树枝瘿姬小蜂体型微小,且无法从形态上区分隐种类型,给该害虫的防治造成困难。本研究基于线粒体DNA细胞色素C氧化酶亚基I（mtDNA COI）基因序列的种特异性（species-specific COI,SS-COI）PCR方法,研究桉树枝瘿姬小蜂隐种快速分子检测技术。基于隐种A、B的COI序列分别设计特异性SS-COI引物各1对（AF/AR和BF/BR）。使用这两对引物扩增同一桉树枝瘿姬小蜂样品DNA,即可有效进行隐种鉴定,同时两对引物也能互相验证鉴定结果。引物鉴定灵敏性检测结果显示,AF/AR与BF/BR均具有较高的鉴定灵敏性,其对DNA的有效鉴定浓度阈值分别为11.42 pg/μL和28.32 pg/μL。本研究开发的桉树枝瘿姬小蜂隐种A、B的快速鉴定方法解决了桉树枝瘿姬小蜂入侵地区隐种鉴别的难题,极大缩短鉴定时间、降低鉴定费用,为进一步探究桉树枝瘿姬小蜂隐种A、B的生物学差异以及它们对不同抗性品种桉树的适应能力提供技术参考。     

Establishment of a mitochondrial DNA sequence database for the identification of fish species commercially available in South Africa

The limitations intrinsic to morphology-based identification systems have created an urgent need for reliable genetic methods that enable the unequivocal recognition of fish species, particularly those that are prone to overexploitation and/or market substitution. The aim of this study was to develop a comprehensive reference library of DNA sequence data to allow the explicit identification of 53 commercially available fish species in South Africa, most of which were locally caught marine species. Sequences of approximately 655 base pairs were generated for all species from the cytochrome c oxidase I (COI) gene, the region widely adopted for DNA barcoding. Specimens of the genus Thunnus were examined in further detail, employing additional mitochondrial DNA control region sequencing. Cumulative analysis of the sequences from the COI region revealed mean conspecific, congeneric and confamilial Kimura 2-parameter distances of 0.10%, 4.58% and 15.43%, respectively. The results showed that the vast majority (98%) of fish species examined could be readily differentiated by their COI barcodes, but that supplementary control region sequencing was more useful for the discrimination of three Thunnus species. Additionally, the analysis of COI data raised the prospect that Thyrsites atun (snoek) could constitute a species pair. The present study has established the necessary genetic information to permit the unambiguous identification of 53 commonly marketed fish species in South Africa, the applications of which hold a plethora of benefits relating to ecology research, fisheries management and control of commercial practices.     

甘肃省鱼类资源现状及DNA条形码在鱼类物种鉴定中的应用

为了摸清甘肃省土著鱼类资源与分布现状, 探索DNA条形码在鱼类辅助物种鉴定中的适用性, 2012年6-9月对甘肃境内黄河水系、嘉陵江水系和河西内陆河水系进行了较全面的鱼类调查。共采集鱼类标本3,087尾, 隶属于5目10科38属64种, 以鲤科种类最多, 为30种, 占总种数的46.88%。物种多样性分析表明, 在黄河水系的夏河和庄浪河多样性指数是所有调查点中最低的, 分别为1.38和1.09。嘉陵江水系各河段的多样性指数较高(H = 2.15-3.27), 其次为河西内陆河水系(H = 2.01-2.83)。在河西内陆河水系中, 疏勒河的均匀度指数最高, 为1.10, 黑河最低(0.68)。庄浪河的优势度指数最高, 为0.34, 而嘉陵江干流两当段的优势度指数在所有调查点中最低, 为0.04。利用DNA条形码分析了49种662尾标本的COI基因部分序列, 大部分种类在neighbor-joining系统树中形成各自的单系, 种内平均遗传距离0.88%, 种间平均遗传距离为9.99%, 在种内和种间COI序列遗传距离之间形成明显的条形码间隙, 斯氏高原鳅(Triplophysa stoliczkae)与达里湖高原鳅(T. dalaica), 甘肃高原鳅(T. robusta)与似鲇高原鳅(T. siluroides), 嘉陵裸裂尻鱼(Schizopygopsis kialingensis)与黄河裸裂尻鱼(S. pylzovi)之间的遗传距离低于2%, 甘肃高原鳅与似鲇高原鳅不能通过COI基因片段区分开, 其他两对物种可以采用核苷酸诊断法来进一步区分。斯氏高原鳅和拉氏鱼岁(Phoxinus lagowskii)种内遗传分歧较大, 揭示种内可能存在隐存种。结果表明, 对某些近缘种和不同地理种群差异较大的物种, 要将分子、形态和地理分布特点结合起来才能准确鉴定。     

Applicability of mitochondrial DNA for the identification of Arvicolid species from faecal samples: a case study from the threatened Cabrera's vole

Arvicolid mitochondrial genomes evolve faster than in any other mammalian lineage. The genetic diversity exhibited by these rodents contrasts sharply with their phenotypic homogeneity. Furthermore, faecal droppings from Arvicolid rodents of similar body size are almost undistinguishable on the basis of pellet morphology and content. In this study, we advantaged from their high genetic diversity vs. phenotypic homogeneity to document the applicability of mtDNA extraction from vole droppings for latter identification of such via a rapid and efficient nested PCR‐based technique using the threatened Microtus cabrerae as a model species. We sequenced the mitochondrial control region from 75 individuals belonging to 11 species of Arvicolinae from Spain, Portugal, Greece and Italy, and an additional 19 sequences from ten Microtus species from other countries were downloaded from Genbank. Based on these control region sequences, we successfully designed and applied a nested PCR for M. cabrerae‐specific and arvicolid‐generic mtDNA markers to differentiate Cabrera’s vole faecal samples among other species of the Arvicolinae subfamily. Although this study used Cabrera’s vole as a model species, similar techniques based on mtDNA sequences may find a broader applicability for noninvasive genetic conservation of vole species and their populations.     

Conserved primers for DNA barcoding historical and modern samples from New Zealand and Antarctic birds

Our ability to DNA barcode the birds of the world is based on the effective amplification and sequencing of a 648 base pair (bp) region of the mitochondrial cytochrome c oxidase (COI or cox1) gene. For many geographic regions the large numbers of vouchered specimens necessary for the construction of a DNA barcoding database have already been collected and are available in museums and other institutions. However, many of these specimens are old (>20 years) and are stored as either fixed study skins or dried skeletons. DNA extracted from such historical samples is typically degraded and, generally, only short DNA fragments can be recovered from such specimens making the recovery of the barcoding region as a single fragment difficult. We report two sets of conserved primers that allow the amplification of the entire DNA barcoding region in either three or five overlapping fragments. These primer sets allow the recovery of DNA barcodes from valuable historical specimens that in many cases are unique in that they are unable or unlikely to be collected again. We also report three new primers that in combination allow the effective amplification from modern samples of the entire DNA barcoding region as a single DNA fragment for 17 orders of Southern Hemisphere birds.     

Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence

Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.     

Co‐amplification of cytochrome b and D‐loop mtDNA fragments for the identification of degraded DNA samples

We propose a simple and effective approach to simultaneously co‐amplify both cytochrome b and D‐loop fragments to evaluate DNA preservation and to monitor possible contaminations in the analysis of degraded animal DNA samples. We have applied this approach to over 200 ancient salmon samples and 25 ancient whale DNA samples, clearly demonstrating its multiple benefits for analysis of degraded DNA samples, and the ease in which co‐amplification can be optimized for different taxa. This simple, cost‐efficient and genomic DNA‐saving approach can be used routinely in the analysis of minute and degraded DNA samples in wildlife forensics, food inspection, conservation biology and ancient faunal remains.     

Species-specific COI primers for rapid identification of Bemisia tabaci Mediterranean (MED) species

Bemisia tabaci (Gennadius) is a rapidly evolving species complex, and is small in size and difficult to identify quickly and accurately. For the accurate identification and effective prevention of this species, the specific PCR method based on the mitochondrial DNA cytochrome oxidase subunit I (mt DNA COI) gene was used in the present study to evaluate rapid molecular detection technological applications for Mediterranean (MED) species. The MED was targeted and whitefly species from different regions were used as references. Fragments of the mt DNA COI gene of the MED and other closely related species were amplified with universal primers. Species-specific mitochondrial DNA cytochrome oxidase subunit I (SS-COI) primers BQLF/BQLR and BQJF/BQJR were designed from variable sites of MED and other whitefly species partial COI gene sequences. Subsequently, the lengths of target fragments were amplified by two pairs of SS-COI primers. Meanwhile, the accuracy, specificity and sensitivity of SS-COI primers were determined using various life stages of the MED and other related species collected from different locations. The primer pairs BQLF/BQLR and BQJF/BQJR generated 334 bp and 483 bp amplified fragment length respectively. Accuracy test results showed that primers can detect the MED single-head adults and also accurately detect single-egg and first instar, second instar and third instar nymphs, MED pupae, etc. Specific detection results demonstrated that the primers were able to amplify the MED but not the following species/populations: Middle East-Asia Minor 1 (MEAM1), Asia I, Asia II 1, Asia II 6 and Asia II 7, Aleurocanthus spiniferus (Quaintanca), A. camelliae, Siphoninus phillyreae, Aleuroclava rhododendri, A. thysanospermi, Aleurolobus taonabae, Dialeurodes citri and Trialeurodes vaporariorum (Westwood) in different areas. Sensitivity detection results showed that primers can detect the minimum threshold of 2,160 pg/μl and 1.38 pg/μl, respectively (equivalent to 1/1280 and 1/2000000 adult). This technique solves the problem that MED cannot be identified based on morphology. This method simultaneously adopted SS-COI PCR technological applications that improved detection accuracy and saved detection time.     

Rapid multiplex PCR based species identification of wild tigers using non-invasive samples

Conservation and management of rare and elusive species requires accurate data on presence or absence. In such cases, molecular genetics based species identification approaches can prove invaluable, especially in conjuncture with non-invasive DNA sampling. However, non-invasive sources yield DNA in low concentration that is degraded, which could result in false negatives for species identification. In this paper, we developed a set of primers for PCR-based species identification of tigers. Our results reveal high rates (upto 90%) of species identification for both fresh (less than 48 h) and old (between 7 days and 3 months) fecal samples from the field. Experiments reveal that multiplex PCR (amplifying more than one genomic region) results in an increase in conclusive species identification (and a consequent decrease in the number of false negatives) from 55% to 89% for old fecal samples. We demonstrate that this increased success is because we experimentally overcome the problems of low DNA template quantity (using the multiplex PCR kit, increases species identification from 55% to 72%) and low template DNA quality (two sets of primers increase the species identification success from 72% to 89%). We recommend that multiplex PCR based methods be used (in conjuncture with species specific primers) for other rare and elusive species since such methods will potentially significantly decrease error in species identification.     

Universal primers for the amplification of chloroplast microsatellites in grasses (Poaceae)

Attempts to design truly universal primers to amplify chloroplast microsatellites have met with limited success due to nonconservation of repeat loci across widely divergent taxa. We have used the complete chloroplast genome sequences of rice, maize and wheat to design five pairs of primers that amplify homologous mononucleotide repeats across the Poaceae (grasses). Sequencing confirmed conservation of repeat motifs across subfamilies and a preliminary study in Anthoxanthum odoratum revealed polymorphism at two loci with a haplotype diversity value of 0.495. These primers provide a valuable tool to study cytoplasmic diversity in this extensively studied and economically important range of taxa.     

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。     

Genotyping of archival Atlantic salmon scales from northern Quebec and West Greenland using novel PCR primers for degraded mtDNA

PCR primers were successfully designed to amplify small ND1 gene fragments for RFLP genotyping of degraded Atlantic salmon Salmo salar mtDNA. Analysis of archival scales with these primers, when existing primer sets failed, show Atlantic salmon from the George River, Quebec, to include European haplotypes and those from the Kapisidlit River, West Greenland, to be fixed for a European haplotype characteristic of Baltic populations.     

New genus-specific primers for the PCR identification of novel isolates of the genus Streptomonospora

The halophilic actinomycete genus Streptomonospora, forming a distinct branch in the 16S rRNA gene phylogenetic tree adjacent to the genera Nocardiopsis and Thermobifida, was first proposed by Cui et al. to accommodate the species type Streptomonospora salina. During a biodiversity and taxonomic study on halophilic filamentous actinomycetes from a saline lake in western China, numerous new halophilic actinomycetes strains were isolated. To confirm whether they are members of the genus Streptomonospora, one set of genus-specific oligonucleotides was designed which allows rapid detection of members of the genus Streptomonospora by means of PCR amplification. The genus specificity of these primers was validated with reference strains as well as with wild-type isolates, which exhibited morphological characteristics common to this genus.     

Unexpected problem in aphid DNA barcoding by universal primers

DNA barcode (mitochondrial COI) sequences have allowed for species identification of aphids. In this study, we newly found a DNA barcoding problem in a part of the DNA sequences for Sitobion avenae. Five S. avenae individuals showed differences of, on average, 32.60% in the DNA sequences from other conspecific individuals, and a BLAST search revealed that the five sequences are similar to those of aphid parasitoids such as Aphidius, Ephedrus and Praon spp. (Hymenoptera: Braconidae). Based on these results, we concluded that the universal primers used in aphid DNA barcodes can amplify barcode sequences from parasitoid species within host aphids.     

Identification of palearctic coregonid fish species using mtDNA and allozyme genetic markers

Two types of molecular genetic markers were used for genetic identification of species and local stocks of palearctic coregonids (Coregonidae, Salmoniformes, Teleostei). Seven nominate species of whitefishes and ciscoes Coregonus , spp. of Eurasia Arctic Sea basin and inconnu Stenodus leucichthys nelma , represented by specimens from North America were studied. Using restriction analysis of PCR-amplified products of the ND-1 gene of mitochondrial DNA (mtDNA) and allelic composition at several allozyme loci discrimination was successful between C. lavaretus pidschian , Siberian whitefish, C. nasus , broad whitefish, C. autumnalis , Arctic cisco, C. migratorius , Baikal omul, C. peled , peled, and C. sardinella , least cisco. Muksun C. muksun , was indistinguishable from Siberian whitefish. Creatine kinase (CK) isozyme patterns and Rsa , I restriction patterns of ND-1 were the most effective markers allowing discrimination among species. Intra-specific differentiation in mtDNA was found in all species but was much less pronounced than inter-species variation. In several specimens composite haplotypes typical of another species were found that reflect probable gene introgression by hybridization. A combination of mtDNA and nuclear genetic markers is suggested for reliable identification of both typical species representatives and hybrids.