Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.     

Simultaneous identification of Grapholita molesta Busck and Grapholita dimorpha Komai by PCR‐RFLP

Although several molecular diagnostic techniques are available for the identification of the apple‐feeding pests Grapholita molesta Busck and Grapholita dimorpha Komai, these pests are severely affecting apple orchards in Korea. These two pests may be misidentified or the available molecular diagnostic techniques may not facilitate the simultaneous identification of the morphological features of both species. In this study, we developed a multiplex assay for these two species using the polymerase chain reaction – restriction fragment length polymorphism (PCR‐RFLP) method. Sixty‐two specimens were collected from apples presumed infested with moth larvae and from pheromone traps from 2013 to 2014. Both species were identified morphologically, and a partial region of the cytochrome b gene was sequenced to design primers for PCR‐RFLP. Digestion profiles of G. molesta and G. dimorpha, using the Sau3A1 restriction enzyme, were characterized using three DNA fragments each for G. molesta (363 bp, 91 bp and 31 bp) and G. dimorpha (220 bp, 234 bp and 31 bp). The RFLP assay developed for both species in this study was more efficient and accurate than other currently used diagnostic assays and would be helpful to identify field‐collected specimens for pest control research.     

Molecular Species Identification of Newly Hatched Hawaiian Amphidromous Gobioid Larvae

This report describes a method for the determination of species identity of newly hatched larvae of five sympatric Hawaiian amphidromous gobioids (Lentipes concolor, Sicyopterus stimpsoni, Awaous guamensis, Stenogobius hawaiiensis, and Eleotris sandwichensis). Polymerase chain reaction (PCR) was used to amplify a homologous section of the cytochrome b (Cyt b) region of the mitochondrial genome (mtDNA) from adults of all five species. The resulting PCR-amplified DNA was subjected to restriction fragment length polymorphism (RFLP) analysis producing species-specific restriction patterns. PCR products from the five species were sequenced to substantiate correct amplification, restriction site locations, and fragment sizes. The sequence data were also used to construct a phylogenetic tree. Individual, newly hatched, wild-caught larvae of amphidromous gobioids of unknown species affinity were sorted into six morphotypes based on physical characteristics. These typed larvae and those from two species that spawned in captivity were subjected to the same molecular analysis as the adults. The RFLP results from adults and larvae were compared, allowing larval morphotypes to be assigned to the appropriate species. These comparisons permitted construction of an identification key to the newly hatched larvae of these species based solely on physical characteristics for use in future field studies. Received April 29, 1998; accepted September 30, 1998.     

Identification of Japanese Lymantria species (Lepidoptera: Lymantriidae) based on PCR–RFLP analysis of mitochondrial DNA

A polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP)-based method for species identification was applied to seven Japanese Lymantria species, including four Asian gypsy moth (AGM) species. We sequenced the partial end of the cytochrome c oxidase I (COI) gene, tRNA leucine, COII gene, and partial end of the tRNA lysine in mitochondrial DNA (mtDNA) for one individual of each of the seven species. We analyzed the recognition sites of three restriction endonucleases and constructed a scheme for Lymantria species identification using PCR–RFLP. We then applied the scheme to 291 individuals from 45 populations of seven species. We found that all seven species were correctly identified using PCR–RFLP. These results suggest that PCR–RFLP is useful for identifying Japanese Lymantria species, which may be detected at Japanese ports.     

A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for <Emphasis Type="Italic">Candida</Emphasis> species identification

Candida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species-specific differentiation of Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of the terminal restriction fragment length polymorphism (t-RFLP) method in the molecular identification of six species of Candida, which are the most common causes of fungal infections. Specific for fungi homocitrate synthase gene was chosen as a molecular target for amplification. The use of three restriction enzymes, DraI, RsaI, and BglII, for amplicon digestion can generate species-specific fluorescence labeled DNA fragment profiles, which can be used to determine the diagnostic algorithm. The designed method can be a cost-efficient high-throughput molecular technique for the identification of six clinically important Candida species.     

Differentiation of non-pylori Helicobacter species based on PCR–restriction fragment length polymorphism of the 23S rRNA gene

Phenotypic identification of non-pylori Helicobacter species has always been problematic and time-consuming in comparison with many other bacteria. We developed a rapid two-step identification assay based on PCR–restriction fragment length polymorphism (PCR–RFLP) analysis of the 23S rRNA gene for differentiating between non-pylori Helicobacter species. A new genus-specific primer pair based on all available complete and partial 23S rRNA sequences of Helicobacter species was designed. In silico restriction analysis of variable regions of the 23S rRNA gene suggested SmaI and HindIII endonucleases would provide a good level of differentiation. Analysis of the obtained 23S rRNA RFLP patterns divided all Helicobacter study strains into three species groups (groups A–C) and 12 unique restriction patterns. Wolinella succinogenes also gave a unique pattern. Our proposed PCR–RFLP method was found to be as a valuable tool for routine identification of non-pylori Helicobacter species from human or animal samples.     

DNA preparation method in eggs, immature stages, and diapausing females of Tetranychus spider mites (Acari: Tetranychidae) for diagnostic PCR-RFLP

A DNA preparation method for diapausing females, immature stages, and eggs of spider mites for diagnostic analyses using restriction fragment length polymorphisms after polymerase chain reaction (PCR-RFLP analysis) of the internal transcribed spacer (ITS) region including 5.8 S of the nucleic ribosomal DNA was developed using Tetranychus urticae Koch as a typical example of tetranychid mites. In diapausing females and deutonymphs, the method of crushing an individual mite with a glass rod was eminently successful and a useful method to obtain the DNA template for PCR. For protonymphs, larvae, and eggs older than 48?h after oviposition at 24?°C, the DNA preparation method for a single nematode, crushing a tiny sample with a filter paper chip, was also successful and a useful method to obtain the DNA template from mite individuals (eggs) for PCR. Consequently, we found a single distinctive band of an amplified DNA fragment after electrophoresis in all developmental stages, and digestion of the PCR product by a restriction endonuclease, DraI, resulted in identical banding patterns being exhibited in all developmental stages. Our findings indicate that PCR-RFLP analysis of the ITS region can be used for species identification of diapausing females, immature stages, and eggs of Tetranychus species.     

Terminal Restriction Fragment Length Polymorphism for Identification of Cryptosporidium Species in Human Feces

Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.     

New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in Soil

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.     

PCR–RFLP to distinguish three Phyllanthus sp., commonly used in herbal medicines

The herbs Phyllanthus amarus, P. fraternus and P. urinaria, are known for their use in preparation of herbal medicines. These herbs grow together and are morphologically indistinguishable. Hence, collections of these herbs for raw drug trade, frequently contain mixture of the three species. Such mixing can result in preparation of herbal drugs with altered efficacy. To prevent this, it is essential to develop reliable methods/markers for accurate identification of species. Keeping this in view, PCR–RFLP (Polymerase Chain Reaction–Restriction Fragment Length Polymorphism) of chloroplast genome regions was carried out, using six pairs of universal primers of chloroplast DNA for amplification, followed by restriction with HinfI and TaqI in P. amarus, P. fraternus and P. urinaria. Distinct restriction patterns for the three species were obtained in all primer pair-restriction enzyme combinations. Since the primers used are conserved over a wide range of plant taxa, PCR–RFLP technique using universal cpDNA primers may be applied to other medicinal/economically important plants, posed with similar problems of identification.     

Morphometric and molecular identification of the female castes of Bombus ignitus and B. ardens (Apidae: Hymenoptera)

Among Korean bumblebees, Bombus ignitus and B. ardens are relatively abundant and important for pollination of wildflowers and agricultural crops. Although the males are easily distinguishable phenotypically, the female castes are difficult to identify from each other. Here we evaluated the value of some morphometric characters in species identification. Also, we developed a polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) to discriminate these similar species. In spite of statistically significant differences of some morphological characters between two species, overlapping quantitative traits hindered accurate identification of the species. However, using 435 bp of COI gene and AluI, BspHI and Earl restriction enzymes allowed molecular identifications of these two species with unique profiles from the digestion by these restriction enzymes. This method can also be applied for older specimens with some morphological characters damaged. We also developed species-specific primers for fast and cost-effective identification of these species.     

Identification of Carnobacterium Species by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR

The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNA^Ala and tRNA^Ile, which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.     

Restriction fragment length polymorphism catalog for molecular identification of Japanese Tetranychus spider mites (Acari: Tetranychidae)

Species identification is a basic issue in biosecurity. Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) is a useful molecular diagnostic tool for species identification. However, the lack of transferability of data has been a serious shortcoming of this method. A RFLP catalog, i.e., a graph of PCR-RFLP patterns expected from sequence data, was devised as a tool to facilitate PCR-RFLP data sharing among laboratories. Twelve species of Tetranychus spider mites have been recorded in Japan to date. In this study, we analyzed DNA sequences of the internal transcribed spacer (ITS) region in nuclear ribosomal DNA of 11 Tetranychus species. For the species identification using PCR-RFLP, we chose six candidates from 131 restriction endonucleases and developed an RFLP catalog of all known Japanese Tetranychus species except Tetranychus neocaledonicus André. The RFLP catalog revealed that most Tetranychus species had diagnostic restriction fragments. The RFLP catalog is transferable and simple molecular diagnostic tool, and it has the ability to add more species and newly found intraspecific variations. Therefore, we believe that the RFLP catalog will contribute to biosecurity as a practical diagnostic tool for species identification of spider mites.     

Discrimination between four Simocephalus species from Slovakia using a PCR-RFLP technique

Planktonic crustaceans are traditionally identified based on morphological and morphometric characters. However, such characters may be hardly distinguishable and often overlap between species. A probability of misidentification is thus relatively high. Molecular techniques may increase the accuracy of identification if appropriate markers are used. Aim of our work was to develop a simple molecular procedure enabling discrimination between four species of Simocephalus occurring in Europe. PCR-RFLP technique proved to be suitable for such discrimination. Within the 709 bp fragment of mitochondrial cytochrome c oxidase subunit 1 gene we found unique combinations of restriction sites of the BbsI and SacI enzymes for Simocephalus vetulus, S. exspinosus, S. serrulatus and S. congener. PCR products of samples from several locations in Slovakia were digested with the two enzymes and electrophoresed on an agarose gel. The restriction patterns were clearly visible and easily distinguishable. This method is applicable for identifying the four species in any life-stage. Considering its simplicity and cost-effectiveness it can be widely used as a diagnostic tool for discriminating between Simocephalus species with overlapping morphologic characters.     

Targeting Single-Nucleotide Polymorphisms in the 18S rRNA Gene To Differentiate Cyclospora Species from Eimeria Species by Multiplex PCR

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3′ end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.     

A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World

The Old World bollworm, Helicoverpa armigera (Hübner), and the corn earworm, H. zea (Boddie), are two of the most important agricultural pests in the world. Diagnosing these two species is difficult—adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP) analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion) to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2) amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae.     

A Rapid Genetic Assay for the Identification of the Most Common Pocillopora damicornis Genetic Lineages on the Great Barrier Reef

Pocillopora damicornis (Linnaeus, 1758; Scleractinia, Pocilloporidae) has recently been found to comprise at least five distinct genetic lineages in Eastern Australia, some of which likely represent cryptic species. Due to similar and plastic gross morphology of these lineages, field identification is often difficult. Here we present a quick, cost effective genetic assay as well as three novel microsatellite markers that distinguish the two most common lineages found on the Great Barrier Reef. The assay is based on PCR amplification of two regions within the mitochondrial putative control region, which show consistent and easily identifiable fragment size differences for the two genetic lineages after Alu1 restriction enzyme digestion of the amplicons.     

A contribution to the subgeneric systematics of Oligoryzomys (Rodentia,Muridae) from Argentina by means of PCR–RFLP patterns of mitochondrial DNA

Subgeneric recognition of rodents of the genus Oligoryzomys from Argentina is specially difficult because morphologic and morphometric criteria to identify species are poorly defined. In order to contribute to the unequivocal identification and geographic distribution of Oligoryzomys species, we studied the restriction fragment length polymorphism (PCR-RFLP) patterns of the D-loop region of the mitochondrial DNA in specimens collected from different regions of Argentina. PCR amplification products of 59 individuals were digested with five restriction enzymes. Patterns obtained were used to produce two phylogenetic consensus trees, one obtained with PAUP and the other with MIX analyses. All specimens from the Patagonia (southern region) clustered together and would correspond to O. longicaudatus. Individuals from the North grouped in four clusters (two of them comprising one individual), supported by high bootstrap values. The one including the 82% of the rodents from the northern region would correspond to the species O. chacoensis.     

RFLP-PCR analysis of the aroA gene as a taxonomic tool for the genus Aeromonas

The aroA gene has been identified as a target in screening for the presence of most Aeromonas species so far described by PCR. Synthetic oligonucleotide primers of 24 and 25 nucleotides were used by PCR assay to amplify a sequence of the aroA gene, which encodes 3-phosphoshikimate-1-carboxyvinyltransferase, a key enzyme of aromatic amino acids and folate biosynthetic pathway. A 1236-bp DNA fragment, representing most of the aroA gene, according to the nucleotide sequence of A. salmonicida, was amplified from all Aeromonas species tested, which represented most of the 14 hybridization groups. HaeII digestion of the 1236-bp fragment generated a restriction fragment length polymorphisms which could be used as a powerful tool for identification of aeromonads to the genus level.     

Highly Specific Culture-Independent Detection of YGNGV Motif-Containing Pediocin-Producing Strains

A novel method based on (1) initial microbiological screening and (2) a highly specific PCR is described for selection of strains expressing YGNGV motif-containing pediocin. Initial screening is carried out using spot on the lawn assay for selection of acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity producing strains. This is followed by highly specific PCR for amplification of 406-bp fragment using forward primer: 5′-tggccaatatcattggtggt-3′ targeting signal peptide sequence of pediocin structural gene and reverse primer: 5′-ctactaacgcttggctggca-3′ encoding N-terminus of immunity gene. The assay was validated with Pediococcus pentosaceus NCDC273 and Pediococcus acidilactici NCDC252 using (1) digestion of amplified 406-bp fragment with HindIII restriction enzyme-producing two restriction fragments of expected sizes (227 and 179 bp), (2) nucleotide sequencing of 406-bp fragment from both strains found these pediocins identical to pediocin PA-1/AcH and (3) identification of both pediocins as pediocin PA-1 at protein level using RP-HPLC. The assay was used for screening six strains (3 pediococci, 2 lactobacilli and an Enterococcus faecium) producing acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity. This resulted in the detection of three new strains (P. pentosaceus NCDC35, E. faecium NCDC124 and Lactobacillus plantarum NCDC20) producing YGNGV motif-containing pediocins.