Design and Evaluation of a Specific Primer Pair for the Diagnosis and Identification of <Emphasis Type="Italic">Acanthamoeba polyphaga</Emphasis>

Random amplified polymorphism DNA (RAPD) is a useful tool for species identification. The obtained band patterns can be used for specific primer pair design that may be useful for species diagnosis. In this study, a distinctive a 962-bp band in A. polyphaga band patterns was found, by using the OPC20 primer (ACTTCGCCAC). The DNA fragment was used to design a specific primer pair that was useful for the identification of different isolates as A. polyphaga species. A case of A. polyphaga in disseminated acanthamoebiasis affecting mesenteric nodes is also reported.     

Random amplified polymorphic DNA profiles as a tool for the identification of Acanthamoeba divionensis

In the present study, we demonstrated the Random Amplified Polymorphism DNA (RAPD) diagnostic validity. In our study, we have analyzed RAPD profiles searching for characteristic and useful bands for Acanthamoeba diagnosis at the species level. We found a distinctive 370-bp band in A. divionensis RAPD patterns, using the OPC14 primer (TGCGTGCTTG) (Operon Technologies, Inc., Alameda, CA). The band specificity was confirmed by hybridization, using it as a probe, against all OPC14 amplifications from 10 different Acanthamoeba species. Once we sequenced this band, we used it to design a specific primer pair which showed positive amplification only in A. divionensis isolates.     

Random Amplified Polymorphic DNA Profiles as a Tool for the Identification of Acanthamoeba divionensis

In the present study, we demonstrated the Random Amplified Polymorphism DNA (RAPD) diagnostic validity. In our study, we have analyzed RAPD profiles searching for characteristic and useful bands for Acanthamoeba diagnosis at the species level. We found a distinctive 370-bp band in A. divionensis RAPD patterns, using the OPC14 primer (TGCGTGCTTG) (Operon Technologies, Inc., Alameda, CA). The band specificity was confirmed by hybridization, using it as a probe, against all OPC14 amplifications from 10 different Acanthamoeba species. Once we sequenced this band, we used it to design a specific primer pair which showed positive amplification only in A. divionensis isolates. RID= ID= <E5>Correspondence to:</E5> A. Ortega-Rivas; <E5>email</E5>: aortega&commat;ull.es Received: 5 August 2002 / Accepted: 23 September 2002     

Species-specific PCR primers for the rapid identification of yeasts of the genus Zygosaccharomyces

Species-specific primer pairs that produce a single band of known product size have been developed for members of the Zygosaccharomyces clade including Zygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces kombuchaensis, Zygosaccharomyces lentus, Zygosaccharomyces machadoi, Zygosaccharomyces mellis and Zygosaccharomyces rouxii. An existing primer pair for the provisional new species Zygosaccharomyces pseudorouxii has been confirmed as specific. The HIS3 gene, encoding imidazole-glycerolphosphate dehydratase, was used as the target gene. This housekeeping gene evolves slowly and is thus well conserved among different isolates, but shows a significant number of base pair changes between even closely related species, sufficient for species-specific primer design. The primers were tested on type and wild strains of the genus Zygosaccharomyces and on members of the Saccharomycetaceae. Sequencing of the D1/D2 region of rDNA was used to confirm the identification of all nonculture collection isolates. This approach used extracted genomic DNA, but in practice, it can be used efficiently with a rapid colony PCR protocol. The method also successfully detected known and new hybrid strains of Z. rouxii and Z. pseudorouxii. The method is rapid, robust and inexpensive. It requires little expertise by the user and is thus useful for preliminary, large-scale screens.     

Acanthamoeba royreba sp. n. from a human tumor cell culture

A new species of Acanthamoeba was isolated from a culture of an established line of human choriocarcinoma cells. The identification of this strain, originally called the Oak Ridge strain, and the establishment of a new species for it were based on morphologic, serologic, and immunochemical studies. In general, the structure of the trophozoite did not differ significantly from that of other species of Acanthamoeba, except that a body which more closely resembled a centriole than material described previously as centriolar satellites was observed in trophozoites examined with the electron microscope. The dimensions of the trophozoite were the smallest among the species of Acanthamoeba. The cyst was typical of the genus, but differed from those of other species by its smaller size and the presence of numerous ostioles. Studies of the Oak Ridge strain by immunofluorescence using antisera developed against the isolate and Acanthamoeba culbertsoni, A. castellanii, A. polyphaga, A. rhysodes, A. astronyxis, and A. palestinensis revealed the antigenic uniqueness of the Oak Ridge strain. It was demonstrated by immunoelectrophoretic analyses of the soluble proteins of the Oak Ridge strain that shared approximately 1/2 of its antigenic structure with A. castellanii and A. culbertsoni. The antigenic differences of the isolate from other species of Acanthamoeba were deduced from comparison of the antigenic constitution of these species and the Oak Ridge strain with A. culbertsoni and A. castellanii. Although the strain was initially recognized by its cytopathogenicity for cultures, it did not produce acute infections in mice after intranasal inoculation of 1 X 10(4) ameba/mouse. The foregoing results constituted the basis for the establishment of the Oak Ridge strain as a new species, A. royreba sp. n., in the genus Acanthamoeba.     

Isoenzyme patterns and phylogenetic relationships in Acanthamoeba spp. isolated from contact lens containers in Korea

In order to refer to the basic information regarding the identification of isolates obtained from a contact lens container in Korea, the isoelectric focusing gel electrophoresis was employed to compare the isoenzyme band patterns among Acanthamoeba spp. including eight isolates and the simple pairwise dissimilarity analysis was carried out. For an alkaline phosphate development, isolate 7 and Acanthamoeba polyphaga showed homologous band patterns, and isolates 1, 2, and 3 showed the same patterns. For lactate dehydrogenase, similar patterns were observed in isolates 2 and 3. Isolates 3 and 5 showed homologous band patterns for malate dehydrogenase and glucose phosphate isomerase. For hexokinase, isolates 4, 7, and A, hatchetti showed the same band patterns. In others, a considerable number of interstrain polymorphisms was observed in nine isoenzyme band patterns. In Acanthamoeba group II, genetic distances among isolates 1, 2, 3, 4, and 5 ranged from 0.104 to 0.200. In comparison to A. castellanii, A. hatchetti, and A. polyphaga, genetic distances of isolates 7 and 8 were 0.254 and 0.219, respectively. In Acanthamoeba group III, including A. culbertsoni, A. healyi, and A. royreba, isolate 6 had genetic distances which ranged from 0.314 to 0.336. Finally, when comparing to the six reference Acanthamoeba, it was possible to classify isolates 1, 2, 3, 4, and 5, as genetically close-related species and as independent species group. Furthermore, isolates 6, 7 and 8 were identified as independent species as well.     

Development of a species-specific AFLP-based SCAR marker for authentication of black muntjac (Muntiacus crinifrons)

Black muntjac is a rare and endangered deer endemic to eastern China. Due to the economic and pharmaceutical value of the meat, antlers and skin, the species has chronically suffered from poaching though it is regarded as the state key protected animal. To provide an effective molecular method for authentication of tissue specimen (such as meat, skin etc.) of the species, we developed a Sequence Characterized Amplified Region (SCAR) derived from a species specific Amplification Fragment Length Polymorphism (AFLP) marker. Initially, a 707-bp species specific DNA fragment of the animal was detected by a pair of AFLP primers (Ep7/Mp8). Subsequently, a species-specific primer pair (P-F/P1-R) was designed based on the specific AFLP fragment sequence, obtaining a 298-bp SCAR for the species. Finally, the reliability of the SCAR primers was verified by two separate PCRs using the designed SCAR primers and a cyt b universal primer pair. As expected, all black muntjac samples presented two bands but the others failed to produce the SCAR by merely showing one band. Our results indicated that the SCAR primers developed in this study may provide a useful tool for forensic authentication of black muntjac samples though further testing with larger sample sizes is warranted.     

Species-specific PCR method for identification of Streptococcus downei

AIMS: To establish a rapid method to differentiate Streptococcus downei and S. sobrinus by multiplex PCR. METHODS AND RESULTS: A PCR primer pair specific to S. downei was designed on the basis of the nucleotide sequence of the dextranase gene of S. downei NCTC 11391T. The primer pair specifically detected S. downei, but none of the other mutans streptococci (16 strains of six species). The PCR procedure was capable of detecting 1 pg of genomic DNA purified from S. downei NCTC 11391 and as few as 14 CFU of S. downei cells. The mixture of primer pairs specific to each S. downei (this study) and S. sobrinus (Igarashi et al. 2000) detected only the strains of these two species among all the mutans streptococcal strains, and concomitantly differentiated the two species by species-specific amplicons of different lengths. CONCLUSIONS: The present PCR method is highly specific to S. downei and is useful for detection and identification of S. downei. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiplex PCR using dextranase gene primers is a useful method for simultaneous detection and differentiation of S. downei and S. sobrinus.     

Comparison of a randomly amplified polymorphic DNA (RAPD) analysis and ATB ID 32C system for identification of clinical isolates of different Candida species

The objective of this work was to compare the usefulness of a randomly amplified polymorphic DNA (RAPD) assay to that of the ATB ID32C kit (bioMérieux, France) for identification of different species of Candida isolated from clinical specimens. The RAPD-PCR patterns obtained with OPE-18 primer for identification of clinical isolates were consistent, and the different independent assays revealed reproduction of the band patterns. RAPD with the OPE-18 primer is a very specific and sensitive method for identification of Candida glabrata, Candida guilliermondii, Candida tropicalis, Candida pelliculosa, Candida albicans, Candida krusei, and Candida lusitaniae.     

酒酒球菌的快速特异性PCR鉴定

以Oenococcus oeni苹果酸-乳酸酶基因(mleA)为目标基因,设计了1对特异性引物PmleaL/PmleaR进行酒酒球菌的快速鉴定研究。结果表明,直接以O.oeni的菌落为模板,通过引物对PmleaL/PmleaR的PCR扩增,可得到mleA基因的特异性条带;用此特异性引物进行供试乳酸菌的PCR鉴定,所有O.oeni菌系均得到特异性条带,而供试的其它种类乳酸菌未扩增出目标带。PmleaL/PmleaR可用于O.oeni的快速PCR鉴定。     

Use of 5´-anchored primers for the enhanced recovery of specific microsatellite markers in Brassica napus L.

Microsatellite markers have assumed great significance in biological research. The isolation and characterisation of microsatellites involves DNA library construction and screening, DNA sequencing, primer design and PCR optimisation. When a microsatellite is situated close to the beginning or end of a cloned fragment, specific primers cannot be designed for one of the flanking sequences, thus hindering the utilisation of such microsatellites as markers. The present approach was to use one 5′-anchored primer complementary to the microsatellite sequence in combination with one specific Cy5- labelled primer with a view to retrieving useful microsatellites, which would otherwise be lost. Six pairs of a 5′ anchored primer and a specific primer were used across a set of 31 Brassica napus winter cultivars and one accession each of five additional Brassica species. Using laser fluorometry a single labelled product was observed after amplification with each of four primer pairs, and one primer pair gave two labelled products. Three products corresponded in size with the products expected if 5′ anchoring was effective, indicating the amplification of locus-specific full-length products including all of the microsatellite repeats. All six primer pairs showed polymorphisms across the Brassica species examined, but only one primer pair showed polymorphisms within B. napus, making it useful for genetic analysis in rapeseed cultivars. The other primer pairs could be useful in studying gene introgression into B. napus or for investigating interspecific crosses involving different Brassica species. Received: 5 August 1999 / Accepted: 1 November 1999     

Evaluation of suitable DNA regions for molecular identification of high value medicinal plants in genus Kaempferia

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Kaempferia (Zingiberaceae). Four primer pairs were evaluated (rbcL, rpoC, trnL and ITS1). It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Thus, the primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL primer pair gave the lowest resolution. Our Bar-HRM developed here would not only be useful for identification of Kaempferia plant specimens lacking essential parts for morphological identification but will be useful for authenticating products in powdered form of a high value medicinal species Kaempferia parviflora, in particular.     

Development of an Acanthamoeba-specific reverse dot-blot and the discovery of a new ribotype

Acanthamoeba is a genus of free-living amoebae, of which some species have been found to cause opportunistic infections in humans. The identification of these amoebae in natural and disease samples is based primarily upon morphological features. While these features are more than adequate for identification to the genus level, they are not useful for species-level identification. This not only leads to difficulty in the diagnosis of infections, but it makes an accurate assessment of the natural distribution of acanthamoebae very difficult to achieve. To improve this situation, a detection method was developed that utilizes both selective polymerase chain reaction amplification and the reverse dot-blot. Oligonucleotides were designed to be specific for the described ribosomal groups (or ribotypes) of Acanthamoeba, as well as one specific for the genus itself. When this method was used to analyze a series of Acanthamoeba cultures from Pakistan, a new ribotype was identified in addition to the detection of the ubiquitously distributed T4 type.     

Re-Definition of the Genus Acanthamoeba with Descriptions of Three Species

SYNOPSIS. Ten strains of Acanthamoeba from freshwater habitats were isolated in clonal cultures. Studies were made of trophic structure, nuclear division, cyst structure, some aspects of cytochemistry, and other characteristics. One strain was identified as A. castellanii (Douglas, 1930), one as A. astronyxis (Ray and Hayes, 1954), and 8 as A. polyphaga (Puschkarew, 1913). Strains of Acanthamoeba isolated by other workers were also examined comparatively.
The pattern of nuclear division in all strains resembled that in metazoan cells, with the exception that centrioles were never found. Trophic amoebae had a PAS-positive surface outline. Cyst walls were strongly PAS-positive and also gave a positive test for cellulose with zinc chloroiodide.
The genus Acanthamoeba Volkonsky, 1931 is re-defined, being distinguished from Hartmannella Alexeieff, 1912, emend. Volkonsky, chiefly by the formation of tapering, hyaline pseudopods (acanthopodia) and by a cyst made up of an ectocyst and a polyhedral or stellate endocyst, with excystment by removal of opercula. Other characteristics found in all strains include a distinctive food cup, the presence of many small refractile globules in the cytoplasm of trophic amoebae, and a cyst wall containing cellulose. The degree of spindle convergence, employed by Volkonsky as a generic criterion, was unusable.
Differential diagnoses based principally on cyst structure are offered for A. castellanii, A. astronyxis , and A. polyphaga. The strain previously called Mayorella palestinensis Reich, 1933 is a distinct species of Acanthamoeba.     

Applicability of RAPD markers on silver-stained polyacrylamide gels to ascertain genetic diversity in Peripatus acacioi (Peripatidae; Onychophora)

RAPD (random amplification of polymorphic DNA) molecular markers can be utilized for analyzing genetic variability in populations for which only a few or no molecular markers are available. They were used in a study of an endangered species, Peripatus acacioi, found in the Tripuí Ecological Station, in Ouro Preto, MG, Brazil. The ecological station was specifically created to protect this velvet worm species, the first of this group found in Brazil. For an initial evaluation of the genetic diversity of this species, DNA samples from the lobopods of four individuals, collected at random, were analyzed using RAPD. Each reaction was run with a different primer (Operon RAPD 10-mer Kits), totaling 13 primers (OPC2, OPC3, OPC4, OPC6, OPC8, OPC10, OPC11, OPL2, OPL7, OPL11, OPL13, OPL18, and OPL19). Due to the low amplification yield, RAPD fragments were separated in polyacrylamide gels and stained with silver nitrate. Numerous bands were observed. Fifty-five of the amplified bands proved to be reproducible, both in terms of presence and intensity. Among these, 27 were variable and 28 were constant. The average number of bands per gel was 4.2. Nine of the 13 primers tested allowed the identification of constant and variable bands among these four individuals. RAPD analysis of genetic variation using silver-stained polyacrylamide gel electrophoresis provided measures of band sharing among the individuals, and therefore could be used in population genetics studies of P. acacioi.     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Membrane carbohydrate characterization of Acanthamoeba astronyxis, A. castellanii and Naegleria fowleri by fluorescein-conjugated lectins

A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.     

DNA-based analysis of planktonic methanotrophs in a stratified lake

1. The assemblage of planktonic methanotrophs in a stratified freshwater lake was investigated. Vertical patterns were analysed by denaturing gradient gel electrophoresis, using the primer pair specific for 16S rRNA genes of type I methanotrophs.
2. The resulting banding patterns could be divided into three distinct groups, and sequenced bands were all related to the Methylobacter species. No amplicon was obtained with the primer pair specific for type II methanotrophs.
3. Cloning analysis of the pmoA gene was performed using samples from three water depths (epilimnion, metalimnion and hypolimnion). The compositions of the clone libraries from the three depths were distinct from each other but all three libraries were dominated by clones related to Methylobacter species.     

Identification and strain differentiation of 'Bacteroides fragilis group' species and Prevotella bivia by PCR fingerprinting

Using single consensus primers of genomic nucleotide sequences, PCR-generated fingerprints were used for identification and differentiation of the Bacteroides fragilis group (B. fragilis, B. thetaiotaomicron, B. ovatus, B. distasonis, B. vulgatus) and Prevotella bivia (B. bivius) by comparing the DNA profiles with those of reference strains from the American Type Culture Collection and German Culture Collection. When primed by a single primer phage M13 core sequence, intra-species specific differences and species-specific bands were detected. Using primers derived from the evolutionarily conserved tRNA gene sequence, species-specific patterns were produced. A computer program, GelManager, was used to analyze the profiles and generate dendrograms. The correlation coefficients determined from the DNA fingerprint profiles of the clinical isolates (using the M13 core primer) fell within a narrow range, reflecting a high level of homology within the species. Based on the dendrograms, strains of one species were clearly differentiated from strains of other species. For comparison, SDS-PAGE analysis of whole cell extracts was also performed to obtain protein band patterns of various strains. Because of the simplicity of the PCR fingerprinting method and the ease of performance of computerized evaluation of data, this technique is a useful method for both species and strain differentiation, as well as for characterization of Bacteroides species and Prevotella bivia.