DNA barcodes and microsatellites: How they complement for species identification in the complex genus Tamarix (Tamaricaceae)

DNA barcoding allows the identification of an organism by comparing the sequence of selected DNA regions (barcodes) with a previously compiled database, and it can be useful for taxonomic identification of species in complex genera, such as Tamarix. Many species of this genus show convergent morphology, which leads to frequent errors in their identification. Highly variable genetic markers, such as microsatellites or short sequence repeats (SSR), could be used to differentiate species where DNA barcodes fail. Here, we tested the ability of both, 5 different marker regions (rbcL, matK, ITS, trnH-psbA, and ycf1), and 14 microsatellites, to properly identify Tamarix species, especially those from the Mediterranean Basin, and compared the pros and cons of the different analytical methods for species identification. DNA barcoding allows the genetic identification of certain species in Tamarix. The two-locus barcodes matK + ITS and ITS + ycf1 were the best-performing combinations, allowing up to 69% and 70%, respectively, correct identification. However, DNA barcoding failed in phylogenetically close groups, such as many Mediterranean species. The use of SSR can aid the identification of species, and the combination of both types of data (DNA barcoding and SSR) improved the success. The combination of data was especially relevant in detecting the presence of hybridization processes, which are common in the genus. However, caution must be exercised when choosing the clustering methods for the SSR datasince different methods can lead to very different results.     

锦葵科植物DNA条形码通用序列的筛选

对锦葵科植物样品的ITS、ITS2、rbcL、matK和psbA-trnH序列进行PCR扩增和测序, 比较各序列的扩增效率、测序成功率、种内和种间变异的差异以及barcoding gap图, 使用BLAST1和Nearest Distance方法评价不同序列的鉴定能力, 进而从这些候选序列中筛选出较适合锦葵科植物鉴别的DNA条形码序列。结果表明, ITS序列在采集的锦葵科植物11个种26个样品中的扩增成功率较高, 其种内、种间变异差异和barcoding gap较ITS2、psbA-trnH及rbcL序列具有更明显的优势, 且纳入60个属316个种共1 228个样品的网上数据后, 其鉴定成功率可达89.9%。psbA-trnH序列的扩增和测序成功率最高, 其鉴定成功率为63.2%, 并能鉴别一些ITS序列无法鉴别的种。实验结果表明, ITS和psbA-trnH是较适合鉴别锦葵科植物的DNA条形码序列组合。     

The utility of internally transcribed spacer 2 DNA sequences of the nuclear ribosomal gene for distinguishing sibling species of Trichogramma

The usefulness of the internally transcribed spacer 2 (ITS2) of the nuclear ribosomal gene complex is tested for providing taxonomic characters to identify Trichogramma species. The ITS2 sequences of a group of sibling species of the T. deion/T. pretiosum complexes were determined. A simple and precise identification key to the species of these assemblages was constructed using as taxonomic characters the size of the ITS2 and the difference in restriction length polymorphism of species with similarly sized ITS2. Individual wasps can be identified by amplification of their ITS2 with general primers, determining the size of the PCR product using standard agarose electrophoresis, followed in some species by a DNA-digestion with a restriction enzyme. Because this system works well for a number of closely related species we are hopeful that similar PCR-based identification can be extended to all species of the genus once their ITS2 sequences have been determined. The advantage of this identification system over the morphology-based system is that non-specialists are able to quickly and cheaply identify individual specimens. In addition, species specific primers were tested for the two most common species of these groups (i.e. T. pretiosum and T. deion). These primers can be used either as a direct identification tool or as a method to confirm the identification using the general key. The phylogeny of this group of wasps was also analyzed based on the ITS2 sequence.     

M-PCR： a powerful method for rapid molecular identification of Trichogramma wasps （Hymenoptera： Trichogrammatidae）

Two species-specific primers were designed depending on ITS2 sequence variation of 37 Trichogramma wasps, and these primers were applied to establish an assay,multiplex PCR (M-PCR), for molecular diagnosis of two important Trichogramma wasps,T. confusum and T. dendrolimi, in China. Multiplex-PCR results showed that only target species produced two PCR products, one product of ITS2 region species-specific amplification and one product of its ITS 1 region universal amplification, but other species produced only one ITS1 universal PCR product. Using this method, the target Trichogramma species can be distinguished from other Trichogramma species. Molecular identification based on M-PCR has particular value over morphological technology and other approaches, such as normal molecular and biochemical methods. Furthermore, because M-PCR assay can avoid false negative results, which frequently happen in PCR reaction, this method will be much more accurate and useful for Trichogramma identification, and can be developed as an easy and rapid diagnostic kit applied in the identification and quality monitoring of Trichogramma mass products both in the factory and in the field. Such an easy and rapid diagnostic kit will be valuable in the application of Trichogramma species as a biological control.     

Molecular identification key for pest species of Scirtothrips (Thysanoptera: Thripidae)

Effective plant quarantine and biological control initiatives require rapid and accurate identification of exotic and potentially invasive taxa that may cause high economic losses or environmental damage. The genus Scirtothrips Shull includes several species that are serious agricultural pests, and, because of their minute size and cryptic behavior, prone to undetected transport through international trade of plant material. Although assigning specimens to the genus Scirtothrips is straightforward using traditional taxonomic methods, identification of species is much more difficult and requires expert knowledge of the genus. Furthermore, the validity of some Scirtothrips species is questionable. Therefore, an easy, accurate, and highly reliable technique is desirable for Scirtothrips identification. Here, we provide a simple molecular key based on the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of nuclear ribosomal DNA. Individual specimens can be identified by amplification of their ITS1 and ITS2 regions with general primers and determining the size of the products by using standard agarose gel electrophoresis, followed in some instances by DNA digestion with the restriction enzymes SacII or PspOM I. The advantage of this identification system is that nonspecialists are able to quickly and cheaply identify individual specimens. Material analyzed for this work was collected in the United States (California), India, South Africa, Kenya, Mexico, Guatemala, Honduras, Nicaragua, Costa Rica, Panama, Australia, New Zealand and Raiatea (Society Islands French Polynesia). We have identified seven pest species with the molecular-based methods described here. It is hoped that this system can be extended to other members of the genus as their ITS1 and ITS2 sequences become available. We also provide molecular confirmation for two new Scirtothrips species, one species from Honduras and one species from New Zealand.     

Candida identification: a journey from conventional to molecular methods in medical mycology

The incidence of Candida infections have increased substantially in recent years due to aggressive use of immunosuppressants among patients. Use of broad-spectrum antibiotics and intravascular catheters in the intensive care unit have also attributed with high risks of candidiasis among immunocompromised patients. Among Candida species, C. albicans accounts for the majority of superficial and systemic infections, usually associated with high morbidity and mortality often caused due to increase in antimicrobial resistance and restricted number of antifungal drugs. Therefore, early detection of candidemia and correct identification of Candida species are indispensable pre-requisites for appropriate therapeutic intervention. Since blood culture based methods lack sensitivity, and species-specific identification by conventional method is time-consuming and often leads to misdiagnosis within closely related species, hence, molecular methods may provide alternative for accurate and rapid identification of Candida species. Although, several molecular approaches have been developed for accurate identification of Candida species but the internal transcribed spacer 1 and 2 (ITS1 and ITS2) regions of the rRNA gene are being used extensively in a variety of formats. Of note, ITS sequencing and PCR–RFLP analysis of ITS region seems to be promising as a rapid, easy, and cost-effective method for identification of Candida species. Here, we review a number of existing techniques ranging from conventional to molecular approaches currently in use for the identification of Candida species. Further, advantages and limitations of these methods are also discussed with respect to their discriminatory power, reproducibility, and ease of performance.     

An assessment of potential diatom "barcode" genes (cox1, rbcL, 18S and ITS rDNA) and their effectiveness in determining relationships in Sellaphora (Bacillariophyta)

Due to limited morphological differentiation, diatoms can be very difficult to identify and cryptic speciation is widespread. There is a need for a narrower species concept if contentious issues such as diatom biodiversities and biogeographies are to be resolved. We assessed the effectiveness of several genes (cox1, rbcL, 18S and ITS rDNA) to distinguish cryptic species within the model 'morphospecies', Sellaphora pupula agg. This is the first time that the suitability of cox1 as an identification tool for diatoms has been assessed. A range of cox1 primers was tested on Sellaphora and various outgroup taxa. Sequences were obtained for 34 isolates belonging to 22 Sellaphora taxa and three others (Pinnularia, Eunotia and Tabularia). Intraspecific divergences ranged from 0 to 5bp (=0.8%) and interspecific levels were at least 18bp (=c. 3%). Cox1 divergence was usually much greater than rbcL divergence and always much more variable than 18S rDNA. ITS rDNA sequences were more variable than cox1, but well-known problems concerning intragenomic variability caution against its use in identification. More information and less sequencing effort mean that cox1 can be a very useful aid in diatom identification. The usefulness of cox1 for determining phylogenetic relationships among Sellaphora species was also assessed and compared to rbcL. Tree topologies were very similar, although support values were generally lower for cox1.     

Phylogenetic reconstruction and DNA barcoding for closely related pine moth species (Dendrolimus) in China with multiple gene markers

Unlike distinct species, closely related species offer a great challenge for phylogeny reconstruction and species identification with DNA barcoding due to their often overlapping genetic variation. We tested a sibling species group of pine moth pests in China with a standard cytochrome c oxidase subunit I (COI) gene and two alternative internal transcribed spacer (ITS) genes (ITS1 and ITS2). Five different phylogenetic/DNA barcoding analysis methods (Maximum likelihood (ML)/Neighbor-joining (NJ), "best close match" (BCM), Minimum distance (MD), and BP-based method (BP)), representing commonly used methodology (tree-based and non-tree based) in the field, were applied to both single-gene and multiple-gene analyses. Our results demonstrated clear reciprocal species monophyly for three relatively distant related species, Dendrolimus superans, D. houi, D. kikuchii, as recovered by both single and multiple genes while the phylogenetic relationship of three closely related species, D. punctatus, D. tabulaeformis, D. spectabilis, could not be resolved with the traditional tree-building methods. Additionally, we find the standard COI barcode outperforms two nuclear ITS genes, whatever the methods used. On average, the COI barcode achieved a success rate of 94.10-97.40%, while ITS1 and ITS2 obtained a success rate of 64.70-81.60%, indicating ITS genes are less suitable for species identification in this case. We propose the use of an overall success rate of species identification that takes both sequencing success and assignation success into account, since species identification success rates with multiple-gene barcoding system were generally overestimated, especially by tree-based methods, where only successfully sequenced DNA sequences were used to construct a phylogenetic tree. Non-tree based methods, such as MD, BCM, and BP approaches, presented advantages over tree-based methods by reporting the overall success rates with statistical significance. In addition, our results indicate that the most closely related species D. punctatus, D. tabulaeformis, and D. spectabilis, may be still in the process of incomplete lineage sorting, with occasional hybridizations occurring among them.     

Identification of internal transcribed spacer sequence motifs in truffles: a first step toward their DNA bar coding

This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (< or = 50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies.     

Assessment of candidate plant DNA barcodes using the Rutaceae family

DNA barcoding is a rapidly developing frontier technology that is gaining worldwide attention.Here,seven regions (psbA-trnH,matK,ycf5,rpoC1,rbcL,ITS2,and ITS) with potential for use as DNA barcodes were tested for their ability to identify 300 samples of 192 species from 72 genera of the family Rutaceae.To evaluate each barcode’s utility for species authentication,PCR amplification efficiency,genetic divergence,and barcoding gaps were assessed.We found that the ITS2 region exhibited the highest inter-specific divergence,and that this was significantly higher than the intra-specific variation in the "DNA barcoding gap" assessment and Wilcoxon two-sample tests.The ITS2 locus had the highest identification efficiency among all tested regions.In a previous study,we found that ITS2 was able to discriminate a wide range of plant taxa,and here we confirmed that ITS2 was also able to discriminate a number of closely related species.Therefore,we propose that ITS2 is a promising candidate barcode for plant species identification.     

Pythium recalcitrans sp. nov. revealed by multigene phylogenetic analysis

A new species of Pythium collected from grapevine roots (Vitis vinifera) in South Africa and roots of common beet (Beta vulgaris) in Majorca, Spain, is described. The phylogenetic position of the new species was investigated by multigene sequence analyses of the internal transcribed spacers (ITS1 and ITS2) of the rDNA region, as well as three other nuclear and three mitochondrial coding genes. Maximum likelihood phylogenetic analyses based on ITS rDNA and concatenated beta-tubulin and cytrochrome c oxidase II alignment place Pythium recalcitrans together with P. sylvaticum and P. intermedium. Pythium recalcitrans sp. nov. is morphologically almost indistinguishable from other Pythium species that only form hyphal swellings in culture. However its species status is justified by the distinctiveness of the DNA sequences in all the genes examined. In culture P. recalcitrans exhibits fast radial growth, abundant spherical to subglobose hyphal swellings but produces no zoosporangia. Sexual structures are not seen in agar media but form in autoclaved grass blades floated on water. Multiple antheridia (1-7) are encountered with most of them diclinous and crook-necked. Oospores are thin-walled and either aplerotic or plerotic. P. recalcitrans was pathogenic to seedlings of Beta vulgaris and Solanum lycopersicum.     

Primers<Emphasis Type="Italic">ITS1, ITS2</Emphasis> and<Emphasis Type="Italic">ITS4</Emphasis> detect the intraspecies variability in the internal transcribed spacers and 5.8S rRNA gene region in clinical isolates of fungi

Restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the internal transcribed spacers (ITS1 and ITS2) was used for examination of 66 isolates belonging to 19 species. Intraspecies variability was found in the examined region of 11 species (Candida albicans, C. catenulata, C. colliculosa, C. glabrata, C. kefyr, C. melinii, C. parapsilosis, C. guillermondii, C. solanii, C. tropicalis, Saccharomyces cerevisiae). Region of ITS-5.8S rDNA was amplified using the primers ITS1 and ITS4. The amplicons were digested by HaeIII, HinfI and CfoI. The recognized intraspecies variability was confirmed in the second step, in which the shorter fragments of this region were amplified using primers ITS1 and ITS2 and analyzed by capillary electrophoresis. Considerable intraspecific variability renders this method unsuitable for species identification, whereas it can be useful for epidemiological tracing of isolates.     

In most Bradyrhizobium groups sequence comparison of 16S-23S rDNA internal transcribed spacer regions corroborates DNA-DNA hybridizations

In an extension of a previous small-scale test to assess the use of 16S-23S rDNA internal transcribed spacer (ITS) sequences for rapid grouping of bradyrhizobia, we have sequenced the ITS region of 32 isolates of Bradyrhizobium that had previously been studied using AFLP and DNA-DNA hybridizations. We also included representatives of Afipia and Rhodopseudomonas. Our results indicate that ITS sequences are very diverse among bradyrhizobia. Nevertheless, for most of the bradyrhizobia, the grouping of ITS sequences was in line with AFLP results and DNA-DNA hybridization data. Strains that have at least 95.5% ITS sequence similarity belong to the same genospecies, i.e. they have more than 60% DNA-DNA hybridization values. The ITS sequences can therefore provide a relatively fast way to guide strain identification and aid selection of the reference groups that should be included in DNA-DNA hybridization experiments for precise genotypic identification. The Bradyrhizobium strains isolated from Aeschynomene species showed a much larger diversity in ITS sequences than other bradyrhizobia, possibly as a result of lateral exchange. The above ITS sequence similarity criterion for genospecies therefore does not apply to them, but they can easily be distinguished from other Bradyrhizobium genospecies because they have a distinct tRNA(ala) gene.     

DNA Barcoding of Economically Important Mushrooms: A Case Study on Lethal Amanitas from China

Some species of the genus Amanita are economically important gourmet mushrooms, while others cause dramatic poisonings or even deaths every year in China and in many other countries. A DNA barcode is a short segment or a combination of short segments of DNA sequences that can distinguish species rapidly and accurately. To establish a standard DNA barcode for poisonous species of Amanita in China, three candidate markers, the large subunit nuclear ribosomal RNA (nLSU), the internal transcribed spacer (ITS), and the translation elongation factor 1 alpha (tef1 α) were tested using the eukaryotic general primers for their feasibility as barcodes to identify seven species of lethal fungi and two species of edible ones which can easily be confused with the lethal ones known from China. In addition, A.phalloides—a European and North American species closely related to one of the seven taxa, A.subjunquillea was also included. PCR amplification and sequencing success rate, intra and inter specific variation and rate of species identification were considered as main criteria for evaluation of the candidate DNA barcodes. Although the nLSU had high PCR and sequencing success rates (100% and 100% respectively), occasional overlapping occurred between the intra and inter specific variations. The PCR amplification and sequencing success rates of ITS were 100% and 85.7% respectively. ITS showed high sequence variation among species group and low variation within a given species. There was a relatively high PCR amplification and sequencing success rate for tef1 α (85.7% and 100% respectively), and its intra and inter specific variation was higher than that of ITS or nLSU. All three candidate markers showed hight species resolution. ITS and tef1 α had a more clearly defined barcode gap than nLSU. Our study showed that the tef1 α and nLSU can be proposed as supplementary barcodes for the genus Amanita, while ITS can be used as a primary barcode marker considering that the ITS region may become a universal barcode marker for the fungal kingdom.     

大型经济真菌的DNA条形码研究——以我国剧毒鹅膏为例

鹅膏属（Amanita）部分物种为重要食用真菌,而另外部分物种则是剧毒的,在我国及其他许多国家,每年都有因误食剧毒鹅膏而导致中毒甚至死亡的事件发生。DNA条形码是用一段或几段短的DNA序列来对物种进行快速、准确鉴定的方法。本研究选取三个候选片段,即核糖体大亚基（nLSU）、内转录间隔区（ITS）和翻译延长因子1-α（tef1-α）,使用真核生物通用引物,测试我国已知的7种剧毒鹅膏及2种易混的可食鹅膏,并将欧美分布的但与黄盖鹅膏（A.subjunquillea）亲缘关系密切的绿盖鹅膏（A.phalloides）纳入分析中。nLSU的PCR扩增和测序成功率均为100%,但种内和种间遗传变异偶有重叠。ITS的PCR扩增和测序成功率分别达到100%和85.7%,且具有高的种间变异和低的种内变异。tef1-α的PCR扩增和测序成功率分别达到85.7%和100%,种间和种内遗传分化均高于ITS和nLSU。三个片段的物种分辨率均较高,但与nLSU相比,ITS和tef1-α具有更为明显的barcode gap。鉴于ITS可能会成为真菌界的通用条码,故建议将ITS作为鹅膏属的核心条形码,tef1 α和nLSU作为该属的辅助条形码。     

Identification of Internal Transcribed Spacer Sequence Motifs in Truffles: a First Step toward Their DNA Bar Coding

This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (≤50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies.     

Comparative study on DNA sequences of ribosomal DNA and cytochrome c oxidase subunit 1 of mitochondrial DNA among five species of gnathostomes

The nucleotide sequences of partial 18S, complete internal transcribed spacer region 1 (ITS1), complete 5.8S, complete ITS2 and partial 28S of ribosomal DNA (rDNA) and cytochrome c oxidase subunit 1 of mitochondrial DNA (MCOI) from five species of gnathostomes (G. spinigerum, G. doloresi, G. nipponicum, G. hispidum and G. binucleatum with the former four species being distributed in Japan and Asia) that cause human gnathostomiasis were compared by direct polymerase chain reaction cycle-sequencing. The nucleotide sequences of each region of the18S (613 bp), 5.8S (158 bp) and 28S (598 bp) rDNA from the five species were almost identical. The ITS1 region was different in length for the five species. The nucleotide sequences of each region of ITS2 and partial MCO1 regions were different among the five species. Therefore, these two regions can be used as genetic markers for identification of worms.     

DNA条形码在中国金合欢属中的应用

根据形态特征难以准确地辨别金合欢属植物,DNA条形码技术提供了一种准确地鉴定物种的方法。本文利用条形码技术对中国金合欢属物种的序列(psbA trnH、matK、rbcL和ITS)及其不同组合进行比较,通过计算种内和种间变异进行barcoding gap分析,运用Wilcoxon秩和检验比较不同序列的变异性,构建系统树。结果表明：4个片段均存在barcoding gap,ITS序列种间变异率较psbA trnH、rbcL和matK序列有明显优势,单片段ITS正确鉴定率最高,ITS+rbcL片段联合条码的正确鉴定率最高,因此我们认为ITS片段或条形码组合ITS+rbcL是金合欢属的快速鉴别最理想的条码。     

Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

Background

The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over.

Methodology/Principal Findings

Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level.

Conclusions

The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.     

Identification of Clinically Relevant Fungi and Prototheca Species by rRNA Gene Sequencing and Multilocus PCR Coupled with Electrospray Ionization Mass Spectrometry

Background

Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse.

Methods

One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5′ end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS.

Results

For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively.

Conclusions

rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.