scholarly journals Polymerase chain reaction and restriction fragment length polymorphism analysis of the ITS2 region for differatiation of Brazilian Biomphalaria intermediate hosts of the Schistosoma mansoni

2004 ◽  
Vol 37 (4) ◽  
pp. 351-353 ◽  
Author(s):  
Teofânia Heloísa Dutra Amorim Vidigal ◽  
Kelly Grace Magalhães ◽  
Omar dos Santos Carvalho
Keyword(s):  
Schistosoma Mansoni ◽  
Restriction Enzymes ◽  
Its2 Region ◽  
Morphological Identification ◽  
Snail Species ◽  
Polymorphism Analysis ◽  
Intermediate Hosts ◽  
Phylogenetic Studies ◽  
Polymerase Chain ◽  
Species Specific

We sequenced the internal transcribed spacer 2 of the ribosomal DNA (ITS2-DNAr) from the three Schistosoma mansoni intermediate hosts in Brazil: Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea. Analysis of a restriction map from those sequences allowed us to select putative restriction enzymes able to identify the snail species under study. Four restriction enzymes were used and HpaII provided simple species-specific profiles easily visualized in polyacrylamide gels. The use of ITS2 is advantageous as it provides a small fragment of 460 bp which may be easily amplified by PCR. In the current work, we showed that the amplification of ITS2-DNAr together with HpaII enzyme restriction is an auxiliary molecular tool for the morphological identification of such snails as well as for taxonomic and phylogenetic studies of neotropical planorbids.

scholarly journals First Report of a Group 16SrII Phytoplasma Infecting Chickpea in Oman

Plant Disease ◽  
2006 ◽  
Vol 90 (7) ◽  
pp. 973-973 ◽  
Author(s):  
N. A. Al-Saady ◽  
A. M. Al-Subhi ◽  
A. Al-Nabhani ◽  
A. J. Khan
Keyword(s):  
Nested Pcr ◽  
Animal Feed ◽  
Restriction Enzymes ◽  
Universal Primers ◽  
Polymorphism Analysis ◽  
Disease Symptoms ◽  
First Report ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Group 2

Chickpea (Cicer arietinum), locally known as “Dungo”, is grown for legume and animal feed mainly in the interior region of Oman. During February 2006, survey samples of chickpea leaves from plants showing yellows disease symptoms that included phyllody and little leaf were collected from the Nizwa Region (175 km south of Muscat). Total nucleic acid was extracted from asymptomatic and symptomatic chickpea leaves using a cetyltrimethylammoniumbromide method with modifications (3). All leaf samples from eight symptomatic plants consistently tested positive using a polymerase chain reaction assay (PCR) with phytoplasma universal primers (P1/P7) that amplify a 1.8-kb phytoplasma rDNA product and followed by nested PCR with R16F2n/R16R2 primers yielding a product of 1.2 kb (2). No PCR products were evident when DNA extracted from healthy plants was used as template. Restriction fragment length polymorphism analysis of nested PCR products by separate digestion with Tru9I, HaeIII, HpaII, AluI, TaqI, HhaI, and RsaI restriction enzymes revealed that a phytoplasma belonging to group 16SrII peanut witches'-broom group (2) was associated with chickpea phyllody and little leaf disease in Oman. Restriction profiles of chickpea phytoplasma were identical with those of alfalfa witches'-broom phytoplasma, a known subgroup 16SrII-B strain (3). To our knowledge, this is the first report of phytoplasma infecting chickpea crops in Oman. References: (1) A. J. Khan et al. Phytopathology, 92:1038, 2002. (2). I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998 (3) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA. 81:8014, 1984.

scholarly journals Large-Restriction-Fragment Polymorphism Analysis of Mycobacterium chelonae and Mycobacterium terrae Isolates

2003 ◽  
Vol 69 (7) ◽  
pp. 4337-4341 ◽  
Author(s):  
J. Daisy Vanitha ◽  
R. Venkatasubramani ◽  
K. Dharmalingam ◽  
C. N. Paramasivan
Keyword(s):  
Restriction Fragment ◽  
Restriction Enzymes ◽  
Computer Assisted ◽  
Polymorphism Analysis ◽  
Chromosomal Dna ◽  
Mycobacterium Chelonae ◽  
Restriction Fragment Polymorphism ◽  
Percent Similarity ◽  
Species Specific ◽  
High Degree

ABSTRACT Mycobacterium chelonae and Mycobacterium terrae were reported to be frequently present in the environment of the Mycobacterium bovis BCG trial area in south India. Six isolates of M. chelonae and four isolates of M. terrae obtained from different sources in this area were analyzed by pulsed-field gel electrophoresis (PFGE) to examine large-restriction-fragment (LRF) polymorphism using the chromosomal DNA digested with DraI and XbaI restriction enzymes. With the exception of one isolate of M. terrae, DNA from all other isolates could be digested with DraI and XbaI and resulted in separable fragments. Visual comparison of the LRFs showed a unique pattern for each of the isolates tested. A computer-assisted dendrogram of the percent similarity demonstrated a high degree of genetic diversity in this group of isolates. This study demonstrates that species of nontuberculous mycobacteria, particularly M. chelonae and M. terrae, can be successfully typed by their LRF pattern using PFGE, which does not require species-specific DNA probes.

Multiplex TaqMan Real-Time PCR Assay for Sensitive Detection of Two Weevil Species (Coleoptera: Curculionidae)

2020 ◽  
Author(s):  
Carlos Aguirre ◽  
Evelyn Sánchez ◽  
Natalia Olivares ◽  
Patricio Hinrichsen
Keyword(s):  
Real Time ◽  
Citrus Fruit ◽  
Sensitive Detection ◽  
Morphological Identification ◽  
Specific Detection ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Cost Efficient ◽  
Species Specific ◽  
Template Dna

Abstract Rapid and cost-efficient identification of Naupactus species is becoming a key process for the exportation of citrus fruit from Chile and other countries, considering the quarantine regulations for some species of the cosmopolitan genus Naupactus. This study deals with the development of a fast and sensitive detection protocol for Naupactus cervinus (Coleoptera: Curculionidae) (Boheman) and Naupactus xanthographus (Coleoptera: Curculionidae) (Germar) based on multiplex TaqMan Real-time polymerase chain reaction. Both N. cervinus and N. xanthographus primer and probe sets achieved species-specific detection in a linear range from 1 pg/μl to 1 × 10-6 pg/μl, allowing detection of as few as 160 copies of template DNA. Non-target amplifications were not detected and a panel composed of 480 test samples had 100% coincidence with the respective morphological identification.

scholarly journals Genetic Diversity Within Colletotrichum acutatum sensu Simmonds

2001 ◽  
Vol 91 (6) ◽  
pp. 586-592 ◽  
Author(s):  
Stanley Freeman ◽  
Dror Minz ◽  
Marcel Maymon ◽  
Aida Zveibil
Keyword(s):  
Sequence Analysis ◽  
Its Region ◽  
Molecular Methods ◽  
Colletotrichum Acutatum ◽  
Morphological Identification ◽  
Chain Reaction ◽  
Subgroup Ii ◽  
Polymerase Chain ◽  
Percent Similarity ◽  
Species Specific

Isolates of Colletotrichum acutatum from several hosts were characterized by various molecular methods in comparison with morphological identification. Species-specific primer analysis was reliable for grouping C. acutatum isolates to their designated species. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses identified four subgroups within C. acutatum. Subgroup I contained U.S. isolates from almond, apple, peach, and pecan, subgroup II contained isolates from anemone, olive, and strawberry, subgroup III contained isolates from almond (Israel) and strawberry (Spain), and subgroup IV contained a single isolate from anemone (the Netherlands). Likewise, sequence analysis of the internal transcribed spacer (ITS) 2 region alone or the complete ITS (ITS 1–5.8S-ITS 2) region grouped the isolates into the same four subgroups. Percent similarity of the complete ITS region within each cluster ranged from 99.6 to 100.0, 99.8 to 100.0, and 98.6% among subgroups I, II, and III, respectively. DNA sequence analysis of the ITS 2 region alone or the entire ITS 1-2 region was more informative than that of the ITS 1 region, which could only group the isolates into two main clusters. The molecular methods employed for studying genetic variation in populations of C. acutatum determined that this species is diverse, indicating that isolates within populations of each subgroup are not host specific.

Genetic variability and molecular identification of Brazilian Biomphalaria species (Mollusca: Planorbidae)

Parasitology ◽  
2001 ◽  
Vol 123 (7) ◽  
pp. 197-209 ◽  
Author(s):  
O. S. CARVALHO ◽  
R. L. CALDEIRA ◽  
A. J. G. SIMPSON ◽  
T. H. D. A. VIDIGAL
Keyword(s):  
Polymerase Chain Reaction ◽  
Morphological Characteristics ◽  
Neotropical Region ◽  
Snail Host ◽  
Molecular Techniques ◽  
Its2 Region ◽  
Intermediate Hosts ◽  
Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain

Freshwater snails belonging to the genus Biomphalaria are intermediate hosts of the trematode Schistosoma mansoni in the Neotropical region and Africa. In Brazil, one subspecies and ten species of Biomphalaria have been identified: B. glabrata, B. tenagophila, B. straminea, B. occidentalis, B. peregrina, B. kuhniana, B. schrammi, B. amazonica, B. oligoza, B. intermedia and B.t. guaibensis. However, only the first three species are found naturally infected with S. mansoni. The classical identification of these planorbids is based on comparison of morphological characteristics of the shell and male and female reproductive organs, which is greatly complicated by the extensive intra-specific variation. Several molecular techniques have been used in studies on the identification, genetic structure as well as phylogenetic relationships between these groups of organisms. Using the randomly amplified polymorphic DNAs (RAPD) analysis we demonstrated that B. glabrata exhibits a remarkable degree of intra-specific polymorphism. Thus, the genetics of the snail host may be more important to the epidemiology of schistosomiasis than those of the parasite itself. Using the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) in intra-populational and intra-specific studies we have demonstrated that snails belonging to the B. straminea complex (B. straminea, B. kuhniana and B. intermedia) clearly presented higher heterogeneity. Using the low stringency polymerase chain reaction (LS-PCR) technique we were able to separate B. glabrata from B. tenagophila and B. tenagophila from B. occidentalis. To separate all Brazilian Biomphalaria species we used the restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer region (ITS) of the DNA gene. The method also proved to be efficient for the specific identification of DNA extracted from snail eggs. Recently we have sequenced the ITS2 region for phylogenetic studies of all Biomphalaria snails from Brazil.

Molecular and morphological discrimination betweenTylospora fibrillosaandTylospora asterophoramycorrhizae

1999 ◽  
Vol 77 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Ursula Eberhardt ◽  
Lutz Walter ◽  
Ingrid Kottke
Keyword(s):  
Fragment Length Polymorphism ◽  
Pcr Primers ◽  
Morphological Identification ◽  
Chain Reaction ◽  
Specific Primers ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Species Specific ◽  
First Time

Among the mycorrhizal types of spruce, Tylospora-type mycorrhizae are the most constant and abundant. Two species of the genus Tylospora occur in Europe, Tylospora fibrillosa and Tylospora asterophora. Mycorrhizae of T. asterophora are described in detail for the first time. Sequences of the internal transcribed spacer (ITS) of the ribosomal genes were obtained from T. fibrillosa and T. asterophora mycorrhizae, sporocarps, and cultured mycelium. Discrimination and identification of the two species by ITS polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) are discussed in the light of inter- and intra-specific variability. Species-specific PCR primers were designed to distinguish both species. Molecular screening of Tylospora-type mycorrhizae from field material led to unambiguous results, whereas morphological identification is likely to fail because of great similarity even at the microscopic level.Key words: Tylospora asterophora, Tylospora fibrillosa, ectomycorrhizae, taxon specific primers (TSOPs), ITS sequences.

scholarly journals A simple PCR-based method for the rapid and accurate identification of spider mites (Tetranychidae) on cassava

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tatiana M. Ovalle ◽  
Aymer Andrés Vásquez-Ordóñez ◽  
Jenyfer Jimenez ◽  
Soroush Parsa ◽  
Wilmer J. Cuellar ◽  
...  
Keyword(s):  
Fragment Length Polymorphism ◽  
Crop Protection ◽  
Restriction Enzymes ◽  
Mite Species ◽  
Morphological Identification ◽  
Chain Reaction ◽  
Accurate Identification ◽  
Polymerase Chain ◽  
Cassava Crop ◽  
Mitochondrial Cytochrome Oxidase

Abstract The morphological identification of mites entails great challenges. Characteristics such as dorsal setae and aedeagus are widely used, but they show variations between populations, and the technique is time consuming and demands specialized taxonomic expertise that is difficult to access. A successful alternative has been to exploit a region of the mitochondrial cytochrome oxidase I (COI) gene to classify specimens to the species level. We analyzed the COI sequences of four mite species associated with cassava and classified them definitively by detailed morphological examinations. We then developed an identification kit based on the restriction fragment length polymorphism–polymerase chain reaction of subunit I of the COI gene focused on the three restriction enzymes AseI, MboII, and ApoI. This set of enzymes permitted the simple, accurate identification of Mononychellus caribbeanae, M. tanajoa, M. mcgregori, and Tetranychus urticae, rapidly and with few resources. This kit could be a vital tool for the surveillance and monitoring of mite pests in cassava crop protection programs in Africa, Asia, and Latin America.

scholarly journals Field Application of NIR Spectroscopy for the Discrimination of the Biomphalaria Species That Are Intermediate Hosts of Schistosoma mansoni in Brazil

2021 ◽  
Vol 9 ◽  
Author(s):  
Vanessa Valladares ◽  
Célio Pasquini ◽  
Silvana C. Thiengo ◽  
Monica A. Fernandez ◽  
Clélia C. Mello-Silva
Keyword(s):  
Schistosoma Mansoni ◽  
Rio De Janeiro ◽  
Near Infrared ◽  
Chemical Components ◽  
Snail Species ◽  
Correct Identification ◽  
Intermediate Hosts ◽  
Simple Method ◽  
Laboratory Populations

Near Infrared Spectroscopy (NIRS) is a spectroscopic technique that evaluates the vibrational energy levels of the chemical bonds of molecules within a wavelength range of 750–2,500 nm. This simple method acquires spectra that provide qualitative and quantitative data on the chemical components of the biomass of living organisms through the interaction between the electromagnetic waves and the sample. NIRS is an innovative, rapid, and non-destructive technique that can contribute to the differentiation of species based on their chemical phenotypes. Chemical profiles were obtained by NIRS from three snail species (Biomphalaria glabrata, Biomphalaria straminea, and Biomphalaria tenagophila) that are intermediate hosts of Schistosoma mansoni in Brazil. The correct identification of these species is important from an epidemiological viewpoint, given that each species has distinct biological and physiological characteristics. The present study aimed to develop a chemometric model for the interspecific and intra-specific classification of the three species, focusing on laboratory and field populations. The data were obtained from 271 live animals, including 150 snails recently collected from the field, with the remainder being raised in the laboratory. Populations were sampled at three localities in the Brazilian state of Rio de Janeiro, in the municipalities of Sumidouro (B. glabrata) and Paracambi (B. straminea), and the borough of Jacarepaguá in the Rio de Janeiro city (B. tenagophila). The chemometric analysis was run in the Unscrambler® software. The intra-specific classification of the field and laboratory populations obtained accuracy rates of 72.5% (B. tenagophila), 77.5% (B. straminea), and 85.0% (B. glabrata). The interspecific differentiation had a hit rate of 75% for the field populations and 80% for the laboratory populations. The results indicate chemical and metabolic differences between populations of the same species from the field and the laboratory. The chemical phenotype, which is closely related to the metabolic profile of the snails, varied between environments. Overall, the NIRS technique proved to be a potentially valuable tool for medical malacology, enabling the systematic discrimination of the Biomphalaria snails that are the intermediate hosts of S. mansoni in Brazil.

scholarly journals Characterization of Colletotrichum Isolates from Tamarillo, Passiflora, and Mango in Colombia and Identification of a Unique Species from the Genus

2003 ◽  
Vol 93 (5) ◽  
pp. 579-587 ◽  
Author(s):  
Lucia Afanador-Kafuri ◽  
Dror Minz ◽  
Marcel Maymon ◽  
Stanley Freeman
Keyword(s):  
Morphological Characterization ◽  
Its2 Region ◽  
Specific Primers ◽  
Molecular Analyses ◽  
Rapd Pcr ◽  
Morphological Criteria ◽  
Polymerase Chain ◽  
Species Specific ◽  
Unique Species

This study was conducted to identify the species of Colletotrichum infecting tamarillo, mango, and passiflora in Colombia and to assess whether cross-infection between host species is occurring. Isolates of Colletotrichum spp. from tamarillo (n = 54), passiflora (n = 26), and mango (n = 15) were characterized by various molecular methods and by morphological criteria. Morphological characterization grouped the tamarillo isolates as C. acutatum and the passiflora and mango isolates as C. gloeosporioides. Species-specific primer analysis was reliable and confirmed grouping of the tamarillo isolates (besides Tom-6) as C. acutatum and the mango isolates (besides Man-76) as C. gloeosporioides. However, DNA of the passiflora isolates was not amplified by either C. acutatum- or C. gloeosporioides-specific primers, but reacted with a new primer, Col1, designed according to the internal transcribed spacer (ITS) 1 region of these isolates. Isolates Tom-6 and Man-76 also reacted positively with the Col1 primer. All the isolates reacting with the C. acutatum- and C. gloeosporioides-specific primers failed to react with primer Col1. Isolate Pass-35 from passiflora did not react with any of the taxon-specific primers. Arbitrarily primed polymerase chain reaction (ap-PCR), random amplified polymerase DNA (RAPD)-PCR, and A+T-rich DNA analyses delineated representative isolates into subgroups within the designated species. Molecular analyses indicated that the C. acutatum tamarillo isolates were uniform or clonal, whereas the C. gloeosporioides mango isolates and Colletotrichum passiflora isolates were heterogeneous. Likewise, sequence analysis of the complete ITS (ITS1-5.8S-ITS2) region identified certain isolates to their respective species: tamarillo isolates as C. acutatum; mango isolates as C. gloeosporioides; passiflora, Tom-6, and Man-76 isolates as a Colletotrichum sp. as yet undefined; and the Pass-35 isolate as an additional undefined Colletot-richum sp. Molecular analyses of the population of Colletotrichum isolates from passiflora, Tom-6 from tamarillo, and Man-76 from mango indicate that this population may not be host specific.

scholarly journals Carnivore Protoparvovirus-1 Associated With an Outbreak of Hemorrhagic Gastroenteritis in Small Indian Civets

2020 ◽  
Vol 57 (5) ◽  
pp. 706-713 ◽  
Author(s):  
Surangkanang Chaiyasak ◽  
Chutchai Piewbang ◽  
Wijit Banlunara ◽  
Somporn Techangamsuwan
Keyword(s):  
Clinical Signs ◽  
Disease Outbreaks ◽  
Disease Spread ◽  
Lymphoid Tissues ◽  
Polymorphism Analysis ◽  
Feline Panleukopenia Virus ◽  
Wildlife Species ◽  
Polymerase Chain ◽  
Generalized Lymphadenopathy ◽  
Species Specific

Carnivore protoparvovirus-1 (CPPV-1) infection has been reported frequently in both domestic and wildlife species including wild carnivores. Fifty-five captive small Indian civets ( Viverricula indica), farmed for perfume production in Eastern Thailand, showed clinical signs of acute bloody diarrhea, anorexia, vomiting, circling, and seizures. The disease spread within the farm and resulted in the death of 38 of the 55 civets (69% mortality) within a month. Fecal swabs were collected from the 17 surviving civets, and necropsy was performed on 7 of the dead civets. Pathologic findings were severe hemorrhagic gastroenteritis with generalized lymphadenopathy. CPPV-1 was identified in both fecal swabs and postmortem samples by species-specific polymerase chain reaction. Further whole-gene sequencing and restriction fragment length polymorphism analysis suggested feline panleukopenia virus (FPV) as the causative agent. The viral tropism and tissue distribution were confirmed by immunohistochemistry, with immunolabeling in the cytoplasm and nucleus of small intestinal crypt epithelial cells, villous enterocytes, histiocytes in lymphoid tissues, myenteric nerve plexuses, and cerebral and cerebellar neurons. Phylogenetic analysis of civet-derived CPPV-1 indicated a genetic similarity close to the FPV HH-1/86 strain detected in a jaguar ( Panthera onca) in China. To our knowledge, this mass die-off of civets is the first evidence of disease associated with CPPV-1 infection in the subfamily Viverrinae. These findings support the multi-host range of parvovirus infection and raises awareness for CPPV-1 disease outbreaks in wildlife species.

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