Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo.     

Comparison of small-scale methods for the rapid extraction of plant DNA suitable for PCR analysis

We present results from a comparison of six methods for rapid DNA extraction from leaf and other plant tissues. We have used samples from six plant species in our study, including both crop species and their wild relatives. The success of the methods is assessed by PCR of the DNA using conserved primers, and the applicability of the different methods to particular species and tissues is assessed. The speed, reliability, convenience, and potential for further improvement of the methods are also discussed.     

Development of compound microsatellite markers in the harmful red tide species Chattonella ovata (Raphidophyceae)

We isolated 12 polymorphic microsatellites from the noxious red‐tide‐causing alga Chattonella ovata. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from four to 12, and the observed and expected heterozygosities ranged from 0.238 to 0.850 and from 0.310 to 0.889, respectively. These loci are useful for revealing the genetic structure of and gene flow among C. ovata populations.     

Development of a Genotyping Method for Potato Scab Pathogens Based on Multiplex PCR

Scab disease significantly damages potato and other root crops. Streptomyces scabiei, S. acidiscabiei, and S. turgidiscabiei are the best-known causal agents of this disease. We have developed a novel genotyping method for these potato scab pathogens using multiplex PCR, whose benefits include rapid and easy detection of multiple species. We designed a species-specific primer set (6 primers, 3 pairs) for the 16S rRNA genes and 16S–23S ITS regions of these potato scab pathogens. The specificity of the primer set was confirmed by testing 18 strains containing potato scab pathogens, other Streptomyces species, and strains of other genera. The application of the developed method to potato field soil and potato tissue samples resulted in the clear detection and identification of pathogens. Since this method is applicable to a large number of environmental samples, it is expected to be useful for a high-throughput analysis of soil and plant tissues of scab disease.     

鼠棒状杆菌PCR检测方法的建立及其应用

目的建立鼠棒状杆菌PCR检测方法并应用于临床样本检测。方法用脑心浸出液培养基复苏、培养鼠棒状杆菌（corynebacteriumkutscheri,C．kutscheri）并提取基因组DNA作模板;根据GenBank中C．kutsche6的16S基因序列设计合成引物,建立鼠棒状杆菌PCR检测方法并进行敏感性和特异性的评价;人工感染昆明鼠,建立小鼠棒状杆菌感染模型,采集肝脏和肾脏,提取DNA进行检测。结果成功建立了鼠棒状杆菌PCR检测方法,该方法可检测到100个阳性质粒;对小鼠沙门氏菌、肺炎链球菌和巴氏杆菌无交叉反应;全部8个人工感染样本全部检测为阳性。结论建立的鼠棒状杆菌PCR检测方法灵敏度高、特异性好,可作为鼠棒状杆菌感染的快速检测方法。     

致病性维罗纳气单胞菌检测方法的建立

发掘维罗纳气单胞菌特异性更强的检测靶点和毒力相关基因靶点,建立能够检测致病性维罗纳气单胞菌的PCR检测方法.通过序列比对分析气单胞菌的16S rRNA基因序列,筛选对维罗纳气单胞菌特异的引物,用于检测种特异性,利用气单胞菌气溶素基因保守引物,检测菌株的致病性,并进行反应条件和反应体系的优化,灵敏度试验和特异性试验.发掘并设计的维罗纳气单胞菌16S rRNA特异性引物结合气单胞菌气溶素基因保守引物建立的检测方法,对12株气单胞菌和10株非气单胞菌的检测结果显示,所有致病性维罗纳气单胞菌都能扩增到大小分别为343 bp和232 bp的特异性条带,而非维罗纳气单胞菌的致病性气单胞菌只能扩增到232 bp的气溶素基因特异性条带,其它菌株都不能扩增到目的条带.灵敏度试验表明,该反应体系的检测灵敏度为1.35×10-3 mg/L.我们建立的致病性维罗纳气单胞菌检测方法能特异地检测致病性维罗纳气单胞菌,并具有高度灵敏性.     

Perkinsus olseni in vitro isolates from the New Zealand clam Austrovenus stutchburyi

Perkinsus olseni infections are reported at 10%-84% prevalences among Austrovenus stutchburyi clams (cockles) in northern New Zealand coastal waters. However, P. olseni has not yet been propagated in vitro from New Zealand clams. In our sample of A. stutchburyi clams from Mangemangaroa Stream, New Zealand, 24% (8/34) showed low-intensity Perkinsus sp. infections among mantle and gill tissues incubated in alternative Ray's fluid thioglycollate medium (ARFTM), and 5% (4/79) showed Perkinsus sp. lesions by histological analyses. Among clams that were screened using a polymerase chain reaction (PCR) assay, 16% (3/19) were positive for Perkinsus sp. DNA. Alternative Ray's fluid thioglycollate medium-enlarged hypnospores from tissues of five infected clams yielded three in vitro Perkinsus sp. isolate cultures that were cloned before sequencing internal transcribed spacer (ITS) regions of their rRNA gene complex. For one isolate, ATCC PRA-205, large subunit (LSU) rRNA and actin genes were also sequenced. All nucleotide sequences from all isolates consistently identified them as P. olseni, as did their in vitro cell cycles and zoosporulation characteristics. All in vitro isolate cultures and their respective monoclonal derivative strains were cryopreserved and deposited for archiving and distribution by the American Type Culture Collection (http://www.atcc.org).     

Development of microsatellite markers in the noxious red tide‐causing algae Heterosigma akashiwo (Raphidophyceae)

We isolated 13 polymorphic microsatellites from the noxious red tide‐causing algae, Heterosigma akashiwo. These loci provide a class of highly variable genetic markers as the number of alleles ranged from three to 12 and the observed and expected heterozygosities ranged from 0.286 to 0.926 and from 0.314 to 0.888, respectively. We consider these loci potentially useful for detailing the genetic structure and gene flow among H. akashiwo populations.     

Development of PCR assay based on ITS2 rDNA polymorphism for the detection and differentiation of Fusarium sporotrichioides

A polymerase chain reaction assay was developed for detection of Fusarium sporotrichioides, a plant pathogen in many parts of the world. Based on small nucleotide differences in ITS2 (Internal Transcribed Spacer) rDNA of our local isolate of F. sporotrichioides (Accession No. AY510069) and other isolates found in NCBI/GeneBank database, species specific primer FspITS2K was selected. Primer pair FspITS2K and P28SL amplified a fragment of 288 bp containing a portion of ITS2 and 28S rDNA of all the F. sporotrichioides isolates tested, originated from different hosts and regions of the world but did not amplify any other species of Fusarium and plant's DNA. To use the PCR assay in seed health testing, a protocol was setup for the rapid and effective preparations of fungal DNA from wheat seeds. The method developed may be useful for the rapid detection and identification of F. sporotrichioides both from culture and from plant tissue.     

Development of an in vitro clonal culture and characterization of the rRNA gene cluster of Perkinsus atlanticus,a protistan parasite of the clam Tapes decussatus

Perkinsus atlanticus cultures were established either with trophozoites isolated from fresh gills, with hypnospores isolated from tissues incubated in fluid thioglycollate medium, or directly from infected hemocytes of carpet shell clams Tapes decussatus from Algarve (Southern Portugal), using a culture medium and conditions optimized for Perkinsus marinus. Perkinsus atlanticus isolates were cloned by limiting dilution, and their identity unequivocally established by PCR-based species-specific diagnostic assays, and by sequencing the complete rRNA gene cluster. The rRNA gene cluster is 7.5-kb in length including 5S, IGS, SSU, ITS1, 5.8S, ITS2, LSU, and an inter-cluster spacer. rDNA sequences of the P. atlanticus clone were between 98.3-100% identical to P. atlanticus sequences previously obtained from clam tissue (non-clonal) isolates. Based on the IGS sequences available from Perkinsus species, a set of primers was designed to amplify P. atlanticus and the two clonally cultured Perkinsus species (P. marinus and P. andrewsi) currently available from a recognized repository. This Perkinsus "genus-specific" PCR-based assay complements the species-specific assays developed earlier and strengthen the detection of Perkinsus species for which specific detection assays are not yet available.     

Improved PCR primers for the detection and identification of arbuscular mycorrhizal fungi

A set of PCR primers that should amplify all subgroups of arbuscular mycorrhizal fungi (AMF, Glomeromycota), but exclude sequences from other organisms, was designed to facilitate rapid detection and identification directly from field-grown plant roots. The small subunit rRNA gene was targeted for the new primers (AML1 and AML2) because phylogenetic relationships among the Glomeromycota are well understood for this gene. Sequence comparisons indicate that the new primers should amplify all published AMF sequences except those from Archaeospora trappei. The specificity of the new primers was tested using 23 different AMF spore morphotypes from trap cultures and Miscanthus sinensis, Glycine max and Panax ginseng roots sampled from the field. Non-AMF DNA of 14 plants, 14 Basidiomycota and 18 Ascomycota was also tested as negative controls. Sequences amplified from roots using the new primers were compared with those obtained using the established NS31 and AM1 primer combination. The new primers have much better specificity and coverage of all known AMF groups.     

Development of a nested PCR detection procedure for Nectria fuckeliana direct from Norway spruce bark extracts

A pair of primers specific for Nectria fuckeliana, a bark infecting pathogen predominantly of Norway spruce (Picea abies), were designed from comparisons of nucleotide sequences of the nuclear ribosomal internal transcribed spacer (ITS) regions of nine isolates from Norway, Lithuania, Switzerland, Austria, Slovakia, Scotland (Larix sp.) and New Zealand (Pinus radiata), and other closely related nectriaceous species, including Neo. Neomacrospora, and 'N'. mammoidea, to which it exhibits taxonomic similarities. Complete ITS sequence homology was observed between each of the nine N. fuckeliana isolates, regardless of geographic provenance, including a previously published Danish strain. Primers Cct1 and Cct2 consistently amplified a single product of 360 bp from DNA prepared from 20 isolates covering the principle range of the disease from Central and Northern Europe, but not from other Neonectria, 'Nectria' or a range of species commonly encountered in forest ecosystems, as well as P. abies or P. radiata DNA. A quick, simple and efficient mechanical lysis procedure for the extraction of high quality total DNA from bark, coupled with post-extraction polyvinylpolypyrrolidone (PVPP) chromatography purification, is described to facilitate successful PCR detection of N. fuckeliana direct from bark extracts. Detection of N. fuckeliana from bark preparations was only possible following nested PCR of PVPP purified extracts using universal primers ITS5 and 4 in first round amplification. The identity of products from bark tissues was confirmed by Southern hybridisation and sequencing. Using the above procedure, positive diagnosis of N. fuckeliana was achievable within 5 h and has the potential for full exploitation as both a forest management and ecological research tool. As the DNA extraction procedure described here has been successful in application against other tree species, it has potential for incorporation into other molecular diagnostic systems for other microorganisms responsible for other wood or tree bark diseases.     

Development of microsatellite markers for the red tide‐forming harmful species Chattonella antiqua,C. marina,and C. ovata (Raphidophyceae)

We have developed 11 microsatellite markers that are specific to Chattonella antiqua, C. marina, and C. ovata, the red tide‐forming harmful phytoplanktons. The 11 loci were amplified in the three species. The number of alleles per locus ranged from 5 to 16. The three species shared most microsatellite regions, although the genetic differences in specific loci were detected among them. These markers of the Chattonella species will be beneficial for biogeographical, detailed taxonomic, studies.     

Delimitation of the Thoracosphaeraceae (Dinophyceae), including the calcareous dinoflagellates, based on large amounts of ribosomal RNA sequence data

The phylogenetic relationships of the Dinophyceae (Alveolata) are not sufficiently resolved at present. The Thoracosphaeraceae (Peridiniales) are the only group of the Alveolata that include members with calcareous coccoid stages; this trait is considered apomorphic. Although the coccoid stage apparently is not calcareous, Bysmatrum has been assigned to the Thoracosphaeraceae based on thecal morphology. We tested the monophyly of the Thoracosphaeraceae using large sets of ribosomal RNA sequence data of the Alveolata including the Dinophyceae. Phylogenetic analyses were performed using Maximum Likelihood and Bayesian approaches. The Thoracosphaeraceae were monophyletic, but included also a number of non-calcareous dinophytes (such as Pentapharsodinium and Pfiesteria) and even parasites (such as Duboscquodinium and Tintinnophagus). Bysmatrum had an isolated and uncertain phylogenetic position outside the Thoracosphaeraceae. The phylogenetic relationships among calcareous dinophytes appear complex, and the assumption of the single origin of the potential to produce calcareous structures is challenged. The application of concatenated ribosomal RNA sequence data may prove promising for phylogenetic reconstructions of the Dinophyceae in future.     

Development of a novel triplex PCR assay for the detection and differentiation of thermophilic species of Campylobacter using 16S-23S rDNA internal transcribed spacer (ITS) region

AIM: Campylobacter species are significantly implicated in human gastrointestinal infections. Of 20 species of Campylobacter, C. jejuni, C. coli and C. lari have been considered as the most important causative agents of human infections. In order to better understand the occurrence and epidemiology of these thermophilic Campylobacter species, an improved and rapid detection method is warranted. A novel triplex polymerase chain reaction (PCR) assay was developed based on the variable 16S-23S rDNA internal transcribed spacer (ITS) region to identify and discriminate between these species in water samples. METHODS AND RESULTS: Campylobacter species-specific primers for C. jejuni, C. coli and C. lari derived from highly variable sequences in the ITS region were used. Specificity of the newly designed primers and PCR conditions were verified using other species of Campylobacter as well as 31 different negative control species. The assay was further validated with 97 Campylobacter cultures from water samples. CONCLUSIONS: The assay was found to be simple, easy to perform, and had a high sensitivity, specificity and reproducibility. It enabled simultaneous detection and differentiation of multiple Campylobacter species in water samples. SIGNIFICANCE AND IMPACT OF STUDY: Use of the newly developed PCR assay, coupled with a previously developed rapid DNA template preparation step, will enable improved detection capabilities for Campylobacter species in environmental matrices.     

RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL‐TIME PCR ASSAYS1

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real‐time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small‐subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.     

实验动物CAR杆菌16SrRNA基因PCR监测方法的建立

目的建立CAR杆菌的PCR监测方法 ,筛查国内部分实验动物样本中CAR杆菌携带状况。方法利用CAR杆菌的特有16SrRNA基因序列片段267bp设计引物,通过从日本实验动物中央研究所获取的CAR标准株DNA,建立实验动物CAR杆菌16SrRNA基因PCR监测方法。结果利用建立的CAR杆菌16SrRNA基因PCR监测方法对国内455份实验动物样本进行筛查,未检出CAR杆菌感染。结论建立了敏感性好,特异性高的实验动物CAR杆菌PCR监测方法 ,未见动物携带CAR杆菌。