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.

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

2021 ◽  
pp. 030098582199156
Author(s):  
Alexandra N. Myers ◽  
Unity Jeffery ◽  
Zachary G. Seyler ◽  
Sara D. Lawhon ◽  
Aline Rodrigues Hoffmann
Keyword(s):  
Polymerase Chain Reaction ◽  
Diagnostic Accuracy ◽  
Molecular Techniques ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Identification Of Fungi ◽  
Fungal Dna ◽  
Polymerase Chain ◽  
Panfungal Pcr ◽  
Pcr Targeting

Molecular techniques are increasingly being applied to stained cytology slides for the diagnosis of neoplastic and infectious diseases. Such techniques for the identification of fungi from stained cytology slides have not yet been evaluated. This study aimed to assess the diagnostic accuracy of direct (without nucleic acid isolation) panfungal polymerase chain reaction (PCR) followed by sequencing for identification of fungi and oomycetes on stained cytology slides from dogs, cats, horses, and other species. Thirty-six cases were identified with cytologically identifiable fungi/oomycetes and concurrent identification via fungal culture or immunoassay. Twenty-nine controls were identified with no cytologically or histologically visible organisms and a concurrent negative fungal culture. Direct PCR targeting the internal transcribed spacer region followed by sequencing was performed on one cytology slide from each case and control, and the sensitivity and specificity of the assay were calculated. The sensitivity of the panfungal PCR assay performed on stained cytology slides was 67% overall, 73% excluding cases with oomycetes, and 86% when considering only slides with abundant fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from control slides with no visible fungus and negative culture results. Direct panfungal PCR is capable of providing genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay should be performed on slides with visible fungi and interpreted in conjunction with morphologic assessment by a clinical pathologist.

scholarly journals Molecular diagnosis and biochemical studies of tick-borne diseases (anaplasmosis and babesiosis) in Aberdeen Angus Cattle in New Valley, Egypt

2020 ◽  
Vol 13 (9) ◽  
pp. 1884-1891
Author(s):  
Nani Nasreldin ◽  
Rania M. Ewida ◽  
Hatem Hamdon ◽  
Yasser F. Elnaker
Keyword(s):  
Oxidative Stress ◽  
Polymerase Chain Reaction ◽  
Molecular Techniques ◽  
Babesia Bovis ◽  
Blood Parasites ◽  
Chain Reaction ◽  
Infected Female ◽  
Angus Cattle ◽  
Polymerase Chain ◽  
Female Cattle

Background and Aim: Anaplasmosis and babesiosis are tick-borne diseases that threaten livestock production with subsequent considerable economic losses. This study was conducted to diagnose Anaplasma and Babesia infection using molecular techniques in imported Aberdeen Angus cattle imported from Uruguay to El-Kharga Oasis in New Valley, Egypt, and to investigate the effects of disease on some serum biochemical and oxidative stress parameters. Materials and Methods: Blood samples were collected from 31 cattle, 21 diseased and ten apparently normal, of varying ages and sex. The blood was used for the preparation of blood smears, polymerase chain reaction assay, and separation of serum for biochemical investigation. The experimental production farm at the Faculty of Agriculture, New Valley University, was infested with ticks and variable clinical manifestations during the period from December 2017 to March 2018. One calf died of a suspected blood parasite infection. Results: The blood film examination revealed infection by blood parasites in 21 samples. Anaplasma marginale and Babesia bovis were identified in 12 and 14 samples, respectively. A total of 14 samples were examined by polymerase chain reaction (PCR) to make these identifications. Biochemical parameters showed significantly elevated serum alanine aminotransferase, aspartate aminotransferase, total bilirubin (T. Bil), and urea in blood from parasite-infected female cattle and male calves compared with controls. Increased serum total protein, globulin, and creatinine were recorded only in infected female cattle. The blood glucose level was significantly decreased in infected female cattle and male calves compared with controls. Furthermore, albumin and albumin/globulin ratio was significantly reduced in the infected female cattle. Oxidative stress profiles of infected animals showed a significant increase in serum nitric oxide and malondialdehyde, and both total antioxidant capacity and reduced glutathione (GSH) were significantly reduced in comparison with control animals. Conclusion: The incidence of A. marginale and B. bovis infection is high in imported Aberdeen Angus cattle in New Valley Province. PCR methods provide a short-term assessment of disease. An extensive epidemiological survey, employing serology together with molecular genetic methods, monitoring of abundance and distribution of tick vectors, availability of vaccination programs, and tracking of animal transport is also needed for control of blood parasites.

Detection of Aspergillus fumigatus by Polymerase Chain Reaction (PCR)

2019 ◽  
Vol 10 (04) ◽  
pp. 655-661
Author(s):  
Zainab H Abood AL-Asadi
Keyword(s):  
Polymerase Chain Reaction ◽  
Aspergillus Fumigatus ◽  
Oligonucleotide Primer ◽  
Rrna Gene ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain ◽  
The Impact

Aspergillosis refers to fungi infections of the respiratory tract caused by Aspergillus species, especially Aspergillus fumigatus. Infection of A. fumigatus was increased in the last few years due to either resistances to antibiotics or the influence of other factors such as other fungal infections. The present study aimed to review the impact of Aspergillus fumigatus in Aspergillosis cases, and study the role of Singleplex PCR for amplification of ITS1, ITS4 of rRNA gene in the detection of fungal isolate. In this study, One hundred sputum samples were collected from patients admitted to the specialize chest and respiratory diseases center / Baghdad who were suffering from respiratory problems. During these studied, molds were isolation and identification based on Conventional method (Direct microscopy by using 10% KOH, and fungal culture was done on Sabouraud Dextrose agar supplemented with chloramphenicol and on Czapek-Dox agar incubated at 37°C and examined for 3-7 days then macroscopic, microscopic examination of the colony by(lactophenol cotton blue stain )and molecular methods by using Polymerase chain reaction (PCR)technique for identification. The 10% KOH examination was positive for 35 cases, while laboratory culturing was positive for 53 cases. Aspergillus sp were isolated from 44(83%) patients; A. fumigatus was isolated in 23 (42. 4%) patients while A. flavus, A. niger, and A. terreus were isolated from 11 (20. 08%), (13. 2%) and 3 (5. 7%) patients respectively, also isolated Penicillium spp. at percentage 1(1. 9%). In this study. The ages of participants ranged from 10-70years with a mean age of 34years, the males were more susceptible to fungal infection, were recorded 35/53 (66. 3), compared to females were 18/53 (33. 96). The infection of fungi was more prevalent in ages 30-40recorded 26(53. 06%) followed by ages 40-50, 13(26. 5), while the lowest infection recorded in the age group 10- 20 years was 2(2. 04%). DNA isolated from twenty-three A. fumigatus isolates was used as a template, and the specific of oligonucleotide primer sequences were used in conventional PCR to detect the presence of internal transcribed spacer region ( ITS) region of the rRNA gene for Aspergillus fumigates. The results of the PCR amplification of the rRNA gene showed that this gene was present in 19 samples out 23 positive samples which isolation with a PCR product size of approximated 385 bp, while 4 samples out 23 positive samples showed negative results for the presence of this gene as indicated by the absence of the PCR products in their relevant lanes. Statistical analysis revealed that the PCR to have a sensitivity of 95. 1% in the detection of Aspergillus fumigatus in Aspergillosis cases. Polymerase chain reaction (PCR) is a rapid, specific, and sensitive method to detect Aspergillus fumigatus in aspergillosis cases of humans.

scholarly journals Sensitive SYBR Green—Real Time PCR for the Detection and Quantitation of Avian Rotavirus A

2018 ◽  
Vol 6 (1) ◽  
pp. 2 ◽  
Author(s):  
David De la Torre ◽  
Claudete Astolfi-Ferreira ◽  
Ruy Chacon ◽  
Antonio Piantino Ferreira
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Sybr Green ◽  
Molecular Techniques ◽  
Economic Losses ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Rotavirus A ◽  
Avian Nephritis Virus ◽  
Polymerase Chain

Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R2 value of 0.999 and a slope of −3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.

Extraction of microsporidial DNA from modified trichrome-stained clinical slides and subsequent species identification using PCR sequencing

Parasitology ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 701-703 ◽  
Author(s):  
K. S. CHAN ◽  
T. H. KOH
Keyword(s):  
Polymerase Chain Reaction ◽  
Species Identification ◽  
Environmental Samples ◽  
Species Level ◽  
Molecular Techniques ◽  
Chain Reaction ◽  
Clinical Specimens ◽  
Polymerase Chain ◽  
Pcr Conditions ◽  
Hiv Negative

SUMMARYMolecular techniques involving polymerase chain reaction (PCR) and sequencing provide a relatively simple and objective means of identifying microsporidia to species level. We modified previously described methods of DNA extraction and PCR conditions for identification of microsporidia from museum slides, clinical specimens and environmental samples and successfully identifiedVittaforma corneaein 11 out of 13 cases of microsporidial infection from used trichrome-stained slides of corneal scrapings from HIV-negative patients with keratoconjunctivitis.

scholarly journals Two Distinct Genotypes of Spissistilus festinus (Say, 1830) (Hemiptera, Membracidae) in the United States Revealed by Phylogenetic and Morphological Analyses

Insects ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 80
Author(s):  
Elizabeth Cieniewicz ◽  
Victoria Poplaski ◽  
Melina Brunelli ◽  
Jason Dombroskie ◽  
Marc Fuchs
Keyword(s):  
United States ◽  
Polymerase Chain Reaction ◽  
Southeastern United States ◽  
Variable Region ◽  
The United States ◽  
Coi Gene ◽  
Its2 Region ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Spissistilus Festinus

Spissistilus festinus (Say, 1830) (Hemiptera: Membracidae) is a frequent pest of leguminous crops in the Southern United States, and a vector of grapevine red blotch virus. There is currently no information on the genetic diversity of S. festinus. In this study, populations of S. festinus were collected in 2015–2017 from various crops and geographic locations in the United States, and fragments of the mitochondrial cytochrome C oxidase 1 (mt-COI) gene and the nuclear internal transcribed spacer 2 (ITS2) region were characterized by polymerase chain reaction and sequencing. Maximum-likelihood and Bayesian analyses of the mt-COI and ITS2 sequences yielded similar phylogenetic tree topologies, revealing two distinct genetic S. festinus lineages with all of the specimens from California comprising one phylogenetic clade, alongside a single GenBank entry from Arizona, and all specimens from the Southeastern United States comprising a statistically-supported distinct clade, regardless of host and year of collection. The mt-COI gene fragment showed up to 10.8% genetic distance between the two phylogenetic clades. These results suggest the existence of two genotypes within S. festinus in the United States. The only distinct morphological trait between the two genotypes was a less elevated pronotum in the representative specimens from California, compared to the representative specimens from the Southeastern United States. Since this phenotypic feature is inconspicuous, a diagnostic polymerase chain reaction targeting a variable region of the mt-COI fragment was developed to reliably distinguish between the specimens of the two genotypes of S. festinus and to facilitate their specific identification.

scholarly journals Portable sequencer in the fight against infectious disease

2019 ◽  
Vol 65 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Arthur Elia Mongan ◽  
Josef Sem Berth Tuda ◽  
Lucky Ronald Runtuwene
Keyword(s):  
Infectious Disease ◽  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Disease Management ◽  
Acid Composition ◽  
Molecular Techniques ◽  
Chain Reaction ◽  
Major Threat ◽  
The World ◽  
Polymerase Chain

Abstract Infectious disease is still a major threat in the world today. Five decades ago, it was considered soon to be eradicated, but the adaptation of pathogens to environmental pressure, such as antimicrobials, encouraged the emergence and reemergence of infectious disease. The fight with infectious disease starts with prevention, diagnosis, and treatment. Diagnosis can be upheld by observing the cause of disease under the microscope or detecting the presence of nucleic acid and proteins of the pathogens. The molecular techniques span from classical polymerase chain reaction (PCR) to sequencing the nucleic acid composition. Here, we are reviewing the works have been undertaken to utilize a portable sequencer, MinION, in various aspects of infectious disease management.

Minimal intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA of lake trout (Salvelinus namaycush)

Genome ◽  
1994 ◽  
Vol 37 (4) ◽  
pp. 664-671 ◽  
Author(s):  
Lang Zhuo ◽  
S. L. Sajdak ◽  
R. B. Phillips
Keyword(s):  
Polymerase Chain Reaction ◽  
Intraspecific Variation ◽  
Ribosomal Dna ◽  
Internal Transcribed Spacer ◽  
Lake Trout ◽  
Intergenic Spacer ◽  
Coding Region ◽  
Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain

Intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA (rDNA) in lake trout was examined by restriction mapping and sequencing of these regions amplified by the polymerase chain reaction. The length of the first internal transcribed spacer region (ITS-1) was 566 bases and the second internal transcribed spacer region (ITS-2) was 368 bases in lake trout. When the 1.4-kb region including the ITS-1, the 5.8S coding region, and the ITS-2 was amplified from 12 individuals from four populations and digested with eight different enzymes only one intraindividual polymorphism was found that occurred in each population. When the amplified ITS-1 region was sequenced from an additional 10 individuals from five populations, no interindividual variation was found in the sequence. A 6-kb portion of the rDNA repeat unit including 1.6 kb of the 18S coding region, the 5′ external spacer region (5′ ETS), and part of the adjacent intergenic spacer was cloned and a restriction map was prepared for these regions in lake trout. No intraspecific variation was found in the region adjacent to the 18S rDNA, which includes the 5′ ETS, although intraspecific and intraindividual length variation was found in the intergenic spacer region 3–6 kb from the 18S. Sequencing of a 609-b segment of the 5′ ETS adjacent to the 18S coding region revealed the presence of two 41-b repeats. The 198-b sequence between the repeats had some similarity to the 18S coding region of other fishes. Primers were designed for amplification of 559 b of the 5′ ETS using the polymerase chain reaction. No intraspecific variation in this region in lake trout was found when the DNA amplified from this region in 12 individuals from four populations was digested with eight restriction enzymes.Key words: ribosomal DNA, internal transcribed spacer regions, 5′ external spacer region, transcribed spacer, lake trout.

scholarly journals Real-Time Polymerase Chain Reaction for One-Hour On-Site Diagnosis of Pierce's Disease of Grape in Early Season Asymptomatic Vines

2002 ◽  
Vol 92 (7) ◽  
pp. 721-728 ◽  
Author(s):  
N. W. Schaad ◽  
D. Opgenorth ◽  
P. Gaush
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Xylella Fastidiosa ◽  
Molecular Techniques ◽  
Plant Diseases ◽  
Pierce's Disease ◽  
Chain Reaction ◽  
Pierce’S Disease ◽  
Polymerase Chain

Molecular-based techniques, such as polymerase chain reaction (PCR), can reduce the time needed for diagnosis of plant diseases when compared with classical isolation and pathogenicity tests. However, molecular techniques still require 2 to 3 days to complete. To the best of our knowledge, we describe for the first time a real-time PCR technique using a portable Smart Cycler for one-hour on-site diagnosis of an asymptomatic plant disease. Pierce's disease (PD) of grape, caused by the fastidious bacterium Xylella fastidiosa, causes serious losses in grapes in California and the southeastern United States. The disease has been difficult to diagnose because typical leaf scorching symptoms do not appear until late (June and after) in the season and the organism is very difficult to isolate early in the season. Sap and samples of macerated chips of secondary xylem from trunks of vines were used in a direct real-time PCR without extraction of DNA. Using two different sets of primers and probe, we diagnosed PD in 7 of 27 vines (26%) from four of six vineyards sampled 10 to 12 days after bud break in Kern, Tulare, and Napa counties of California. The diagnosis was confirmed by isolation of Xylella fastidiosa from two of the original PCR positive samples and later from symptomatic leaf petioles of four out of four vines from one vineyard that were originally PCR positive.

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