scholarly journals Failure To Differentiate Cryptosporidium parvum from C. meleagridis Based on PCR Amplification of Eight DNA Sequences

1998 ◽  
Vol 64 (4) ◽  
pp. 1454-1458 ◽  
Author(s):  
Dominique Champliaud ◽  
Philippe Gobet ◽  
Muriel Naciri ◽  
Odile Vagner ◽  
José Lopez ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Dna Sequences ◽  
Target Genes ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Restriction Enzyme Digestion ◽  
Human Pathogens

ABSTRACT In order to determine the specificities of PCR-based assays used for detecting Cryptosporidium parvum DNA, eight pairs of previously described PCR primers targeting six distinct regions of theCryptosporidium genome were evaluated for the detection ofC. parvum, the agent of human cryptosporidiosis, andC. muris, C. baileyi, and C. meleagridis, three Cryptosporidium species that infect birds or mammals but are not considered to be human pathogens. The four Cryptosporidium species were divided into two groups: C. parvum and C. meleagridis, which gave the same-sized fragments with all the reactions, and C. muris and C. baileyi, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification, C. parvum and C. meleagridiscouldn’t be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen C. parvum.

scholarly journals Target enrichment from a DNA mixture by oligoribonucleotide interference-PCR (ORNi-PCR)

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Toshitsugu Fujita ◽  
Daisuke Motooka ◽  
Hodaka Fujii
Keyword(s):  
16S Rrna ◽  
Genome Editing ◽  
Dna Sequences ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Detailed Assessment ◽  
Dna Mixture

Abstract Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method that suppresses PCR amplification of target DNA in an ORN-specific manner. In this study, we examined whether ORNi-PCR can be used to enrich desirable DNA sequences from a DNA mixture by suppressing undesirable DNA amplification. ORNi-PCR enriched edited DNA sequences from a mixture of genomic DNA subjected to genome editing. ORNi-PCR enabled more efficient analysis of the types of insertion/deletion mutations introduced by genome editing. In addition, ORNi-PCR reduced the detection of 16S ribosomal RNA (16S rRNA) genes in 16S rRNA gene-based microbiome profiling, which might permit a more detailed assessment of populations of other 16S rRNA genes. Enrichment of desirable DNA sequences by ORNi-PCR may be useful in molecular biology, medical diagnosis, and other fields.

scholarly journals Molecular Characterization of Ciliate Diversity in Stream Biofilms

2008 ◽  
Vol 74 (6) ◽  
pp. 1740-1747 ◽  
Author(s):  
Andrew Dopheide ◽  
Gavin Lear ◽  
Rebecca Stott ◽  
Gillian Lewis
Keyword(s):  
18S Rrna ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Sequence Diversity ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Specific Pcr ◽  
Stream Biofilms

ABSTRACT Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

scholarly journals Quantitative Measure of Small-Subunit rRNA Gene Sequences of the Kingdom Korarchaeota

1998 ◽  
Vol 64 (12) ◽  
pp. 5064-5066 ◽  
Author(s):  
Clifford F. Brunk ◽  
Nicole Eis
Keyword(s):  
Internal Standard ◽  
Pcr Amplification ◽  
Quantitative Measure ◽  
Pcr Primers ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Rdna Sequence ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Rdna Sequences

ABSTRACT Comparative PCR amplification of small-subunit (SSU) rRNA gene (rDNA) sequences indicates substantial preferential PCR amplification of pJP27 sequences with korarchaeote-specific PCR primers. The coamplification of a modified SSU rDNA sequence can be used as an internal standard to determine the amount of a specific SSU rDNA sequence.

scholarly journals Confirmatory Evidence from 18S rRNA Gene Analysis for In Vivo Development of Propamidine Resistance in a Temporal Series ofAcanthamoeba Ocular Isolates from a Patient

1998 ◽  
Vol 42 (8) ◽  
pp. 2144-2145 ◽  
Author(s):  
Dolena R. Ledee ◽  
David V. Seal ◽  
Thomas J. Byers
Keyword(s):  
Mixed Infection ◽  
Dna Sequences ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Gene Analysis ◽  
Rrna Gene ◽  
Temporal Series ◽  
Before And After

ABSTRACT DNA sequences of three 18S rRNA gene alleles present in trophozoites obtained before and after therapy forAcanthamoeba keratitis substantiate a previous report that the infection was due to a single Acanthamoeba strain. Thus, the possibility that propamidine resistance which developed during therapy was due to a mixed infection was ruled out.

scholarly journals Genotyping of Acantamoeba spp. from rhisophere in Hungary

2020 ◽  
Vol 67 (3) ◽  
pp. 171-175
Author(s):  
Erika Orosz ◽  
Katalin Posta
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Human Pathogens ◽  
Quantitative Real Time Pcr ◽  
Potential Health ◽  
Granulomatous Amoebic Encephalitis ◽  
Related Genotype ◽  
Biological Methods

The protista Acanthamoeba is a free-living amoeba existing in various environments. A number of species among protista are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK), granulomatous amoebic encephalitis (GAE), and chronic granulomatous lesions. In this study, 10 rhizosphere samples were collected from maize and alfalfa plants in experimental station at Institute of Genetics, Microbiology and Biotechnology, Szent István University. We detected Acanthamoeba based on the quantitative real-time PCR assay and sequence analysis of the 18S rRNA gene. All studied molecular biological methods are suitable for the detection of Acanthamoeba infection in humans. The quantitative real-time PCR-based methods are more sensitive, simple, and easy to perform; moreover, these are opening avenue to detect the effect of number of parasites on human disease. Acanthamoeba species were detected in five (5/10; 50%) samples. All Acanthamoeba strains belonged to T4 genotype, the main AK-related genotype worldwide. Our result confirmed Acanthamoeba strains in rhizosphere that should be considered as a potential health risk associated with human activities in the environment.

scholarly journals Legionella Species Diversity in an Acidic Biofilm Community in Yellowstone National Park

2005 ◽  
Vol 71 (1) ◽  
pp. 507-511 ◽  
Author(s):  
Kathy B. Sheehan ◽  
Joan M. Henson ◽  
Michael J. Ferris
Keyword(s):  
Yellowstone National Park ◽  
National Park ◽  
Pcr Amplification ◽  
18S Rrna Gene ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Thermal Gradients ◽  
Legionella Species ◽  
Legionella Micdadei

ABSTRACT Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30�C) and higher (35 to 38�C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.

scholarly journals Molecular Assay Proves the Presence of Theileria annulata Infection in Camels in Al-Diwaniyah Province, Iraq

2021 ◽  
Author(s):  
Noaman N. A'aiz ◽  
Hayder N. Ayyez ◽  
Ahmed J. Neamah
Keyword(s):  
Dna Amplification ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Molecular Assay ◽  
Conventional Pcr ◽  
Molecular Investigation ◽  
Causative Agents ◽  
The One ◽  
Specific Species ◽  
Theileria Spp

Background: Theileria camelensis and T. dromedarii are parasitic protozoans reported by several studies as specific species that infect the one-humped camel (Camelus dromedarius). However, other findings casted significant doubts on the true identity of the causative species of theileriosis in camels. Therefore, the present study was conducted to investigate of T. camelensis and T. dromedarii in one humped camels in Iraq during Apr-Oct 2017. Methods: Blood samples for DNA extraction were obtained from 181 slaughtered camels. Molecular investigation was performed following the amplification of 18S rRNA gene by conventional PCR technique. DNA sequencing was then utilized only for the positive samples to confirm the infection with the Theileria species. Results: Nine (4.97%) out of 181 examined samples showed a positive result to infection with Theileria spp., and all these appeared as a T. annulata when subjected to DNA amplification and sequencing techniques. There was a complete absence of any new sequence outside the known species. Conclusion: Most of Theileria infection in camels in the study area is caused by T. annulata and no other causative agents like T. camelensis or T. dromedarii.  

scholarly journals Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil

2000 ◽  
Vol 66 (2) ◽  
pp. 844-849 ◽  
Author(s):  
G. Sabat ◽  
P. Rose ◽  
W. J. Hickey ◽  
J. M. Harkin
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Enteric Bacteria ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Selective Amplification ◽  
E Coli

ABSTRACT A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminatingE. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72°C, which is 10°C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl2 concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detectingE. coli DNA in heterogeneous DNA samples, such as those extracted from soil.

Tetractinomyxon stages genetically consistent withSphaerospora dicentrarchi(Myxozoa: Sphaerosporidae) found inCapitellasp. (Polychaeta: Capitellidae) suggest potential role of marine polychaetes in parasite's life cycle

Parasitology ◽  
2016 ◽  
Vol 143 (8) ◽  
pp. 1067-1073 ◽  
Author(s):  
LUIS F. RANGEL ◽  
RICARDO CASTRO ◽  
SÓNIA ROCHA ◽  
RICARDO SEVERINO ◽  
GRAÇA CASAL ◽  
...  
Keyword(s):  
Life Cycle ◽  
Dna Sequences ◽  
Dicentrarchus Labrax ◽  
18S Rrna Gene ◽  
Life Cycles ◽  
Fish Farm ◽  
Rrna Gene ◽  
Commercial Fish ◽  
European Seabass

SUMMARYKnown life cycles of myxosporean parasites have two hosts, but very few life cycles have been disclosed, especially in the marine environment.Sphaerospora dicentrarchiSitjà-Bobadilla and Álvarez-Pellitero, 1992 is a systemic parasite from the European seabass,Dicentrarchus labrax(Linnaeus, 1758), a highly valuable commercial fish. It affects its health, leading to aquaculture production losses. During 2013 and 2014, an actinospore survey was conducted in a total of 5942 annelids collected from a fish farm in Algarve and from the Aveiro Estuary, in Portugal. A new tetractinomyxon actinospore was found in a capitellid polychaete, belonging to the generaCapitellacollected at the fish farm. The tetractinomyxons were pyriform measuring 11·1 ± 0·7µm in length and 7·2 ± 0·4µm in width, and presented three rounded polar capsules measuring 2·4 ± 0·3µm in diameter. The molecular analysis of the 18S rRNA gene sequences from the tetractinomyxons revealed a similarity of 100% with the DNA sequences deposited in the GenBank fromS. dicentrarchimyxospores collected from the European seabass and the spotted seabass in the same fish farm and 99·9% similarity with the DNA sequence obtained from the myxospores found infecting the European seabass in the Aveiro Estuary. Therefore, the new tetractinomyxons are inferred to represent the actinospore phase of theS. dicentrarchilife cycle.

DNA AMPLIFICATION OPTIMIZATION AND CLONING OF SEVERAL TARGET GENES FROM BURKHOLDERIA PSEUDOMALLEI

2015 ◽  
Vol 77 (25) ◽  
Author(s):  
Abdul Latif Osman ◽  
Syed Mohamed Syed Abdullah ◽  
Siti Marhamah Drahaman ◽  
Mohd Firdaus Raih ◽  
Noraslinda Muhamad Bunnori ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Target Genes ◽  
Latent Infection ◽  
Insertion Site ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Hypothetical Proteins ◽  
Northern Australia ◽  
Expression And Purification ◽  
Protein Targets

Burkholderia pseudomallei is a saprophytic Gram-negative bacillus that is found in soil and surface water. It causes the disease melioidosis, which infect humans and animals. Melioidosis can be fatal in human, where it causes fever and commonly present with pneumonia, with or without septicaemia. Melioidosis is primarily endemic in Southeast Asia and Northern Australia, but has also been found in the Middle East, China and South America. It is challenging to treat melioidosis, as it is intrinsically resistant to many antibiotics, and can cause latent infection. By characterizing identified protein targets from B. pseudomallei, we can gain fundamental knowledge on how this bacterium behaves, and thus provide us strategies to combat them. We report here the recent progress of DNA amplification and cloning of four target genes from B. pseudomallei strain D286 performed in Kulliyyah of Science, IIUM. Genomic DNA of B. pseudomallei strain D286 is obtained from School of Biosciences & Biotechnology, UKM. The four target genes; BPSL1612, BPSL1618, BPSL1691 and BPSL2054 were chosen from 52 hypothetical proteins predicted to be essential in B. pseudomallei by transposon-directed insertion site sequencing (TraDIS) technique (Moule et al., 2014). All four target genes were subjected to nested PCR amplification for subsequent GatewayTM cloning protocols, expression and purification studies.

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