scholarly journals From 18S to 28S rRNA Gene: An Improved Targeted Sarcocystidae PCR Amplification, Species Identification with Long DNA Sequences

2021 ◽  
Vol 104 (4) ◽  
pp. 1388-1393
Author(s):  
Florence C. H. Lee ◽  
Vickneshwaran Muthu
Keyword(s):  
Species Identification ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Environmental Samples ◽  
Pcr Amplification ◽  
Rrna Genes ◽  
Rrna Gene ◽  
28S Rrna ◽  
Index Test ◽  
Identity Matching

ABSTRACTSarcocystosis outbreaks in Tioman and Pangkor islands of Malaysia between 2011 and 2014 have raised the need to improve Sarcocystis species detection from environmental samples. In-house works found that published primers amplifying the 18S rRNA gene of Sarcocystis either could not produce the target from environmental samples or produced Sarcocystis DNA sequence that was insufficient for species identification. Using the primer pair of 18S S5 F (published) and 28S R6 R (new), this study improved the PCR amplification of Sarcocystidae to overcome these two difficulties. The PCR product spanned from the 18S to 28S rRNA genes, providing more information for species identification. The long DNA sequence allowed comparison between the “Ident” and “Query Cover” sorting in GenBank identity matching. This revealed the ambiguity in identity matching caused by different lengths of reference DNA sequences, which is seldom discussed in the literature. Using the disparity index test, a measurement of homogeneity in nucleotide substitution pattern, it is shown that the internal transcribed spacer (ITS)1-5.8S-ITS2 and 28S genes are better than the 18S gene in indicating nucleotide variations, implying better potentials for species identification. The example given by the handful of Sarcocystidae long DNA sequences reported herein calls for the need to report DNA sequence from the 18S to the 28S rRNA genes for species identification, especially among emerging pathogens. DNA sequence reporting should include the hypervariable 5.8S and ITS2 regions where applicable, and not be limited to single gene, per the current general trend.

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 identification of parasitoid, Encarsia formosa gahan in Bemisia tabaci (Gennadius) and determination of its secondary endosymbionts

2015 ◽  
Vol 39 (4) ◽  
pp. 563-578
Author(s):  
SMH Jahan ◽  
KY Lee ◽  
MIA Howlader ◽  
HM Bashar ◽  
GN Hasan
Keyword(s):  
Research Work ◽  
Pcr Amplification ◽  
Rrna Genes ◽  
Gene Region ◽  
Molecular Technique ◽  
Rrna Gene ◽  
28S Rrna ◽  
Encarsia Formosa ◽  
Molecular Physiology

In this study two pairs of primers based on mitochondrial cytochrome oxidase subunit 1 (mtCOI) region and 28S ribosomal RNA (rRNA) gene region were used for identifying very tiny and morphologically indistinguishable parasitoid Encarsia formosa (Gahan) which are specific to this insect. The fragment amplified by these primer pairs were 860 and 650 bp in length. Species specificity test showed that all E. formosa specimens were detected with no cross reactions with other aphelinid species, including E. sophia (Girault & Dodd), E. luteola, E. Inaron and E. Nigricephala. Using phylogenetic cladogram by the sequences analysis of both mtCOI and 28S rRNA genes could be detected in E. formosa accurately in all replicates. Cardinium and Wolbachia secondary endosymbiont were also detected in E. Formosa used by PCR amplification as well as sequence analysis of 16S-23S rDNA gene region. The molecular technique developed here would be useful for rapid and precise species identification, determination of the host spectrum and more effective utilization of E. formosa. This research work has been performed from January 2011 to June 2012 at the insect molecular physiology lab in the Republic of Korea. DOI: http://dx.doi.org/10.3329/bjar.v39i4.22532 Bangladesh J. Agril. Res. 39(4): 563-578, December 2014

5S ribosomal and U1 small nuclear RNA genes: A new linkage type in the genome of a crustacean that has three different tandemly repeated units containing 5S ribosomal DNA sequences

Genome ◽  
2001 ◽  
Vol 44 (3) ◽  
pp. 331-335 ◽  
Author(s):  
Franca Pelliccia ◽  
Rita Barzotti ◽  
Elisabetta Bucciarelli ◽  
Angela Rocchi
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
5S Rdna ◽  
5S Rrna ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Small Nuclear Rna ◽  
5S Rrna Gene ◽  
Nuclear Rna ◽  
Pcr Products

We investigated the 5S ribosomal RNA (rRNA) genes of the isopod crustacean Asellus aquaticus. Using PCR amplification, three different tandemly repeated units containing 5S rDNA were identified. Two of the three sequences were cloned and sequenced. One of them was 1842 bp and presented a 5S rRNA gene and a U1 small nuclear RNA (snRNA) gene. This type of linkage had never been observed before. The other repeat consisted of 477 bp and contained only an incomplete 5S rRNA gene lacking the first eight nucleotides and a spacer sequence. The third sequence was 6553 bp long and contained a 5S rRNA gene and the four core histone genes. The PCR products were used as probes in fluorescent in situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus. The possible evolutionary origin of the three repeated units is discussed.Key words: Asellus, isopoda, crustacea, 5S rDNA, U1 snDNA.

Six new species of Heteropriapulus (Monogenea: Dactylogyridae) from South American fishes with an amended diagnosis to the genus

Zootaxa ◽  
2017 ◽  
Vol 4290 (3) ◽  
pp. 459 ◽  
Author(s):  
ALINE ANGELINA ACOSTA ◽  
LIDIANE FRANCESCHINI ◽  
ALINE CRISTINA ZAGO ◽  
TOMÁŠ SCHOLZ ◽  
REINALDO JOSÉ DA SILVA
Keyword(s):  
New Species ◽  
Species Identification ◽  
Type Species ◽  
Accessory Piece ◽  
Molecular Data ◽  
Fish Host ◽  
Rrna Gene ◽  
28S Rrna ◽  
South American ◽  
Unique Shape

Heteropriapulus Kritsky, 2007 (Monogenea: Dactylogyridae), which originally included only two species from the gills of loricariid catfishes, is reviewed and six newly described species from loricariids in the Paraná River basin in Brazil are added. Diagnosis of the genus is amended and a key to the species identification is provided. Heteropriapulus anchoradiatus n. sp. from Pterygoplichthys ambrosettii (Holmberg) (Hypostominae) differs from its congeners by having a long sclerotized vagina, ventral anchors with short shaft and conspicuous superficial root, and a conspicuous and robust postero-medial process on the dorsal bar; H. bitomus n. sp. from the same fish host differs by the presence of two pairs of sclerotized patches associated with the ventral anchors; H. falxus n. sp. from Hypostomus strigaticeps (Regan) (Hypostominae) and Hypostomus ancistroides (Ihering) (Hypostominae) is unique by the shape of the accessory piece composed of two strongly sclerotized subunits; H. microcleithrus n. sp. from P. ambrosettii differs by presenting the smallest length of the dorsal bar and unique shape of the longer subunit of the accessory piece resembling the ‘hammer and sickle’ shape; H. pterygoplichthyi n. sp. from the same host presents unique shape of the longer subunit of the accessory piece of the cirrus, which is represented by ‘two sickles’ jointed by the base; and H. semitortus n. sp. from Rhinelepis aspera Spix & Agassiz (Rhinelepinae) can be distinguished by the accessory piece composed of a single straight unit and a cirrus tube with the highest number of spiral rings at the proximal end (2½). First molecular data for this genus (partial sequences of the 28S rRNA gene) are provided including the type species H. heterotylus (Jogunoori, Kritsky & Venkatanarasaiah, 2004).  

scholarly journals The differential expression of ribosomal 18S RNA paralog genes from the chaetognath Spadella cephaloptera

2007 ◽  
Vol 12 (4) ◽  
Author(s):  
Roxane-Marie Barthélémy ◽  
Michel Grino ◽  
Pierre Pontarotti ◽  
Jean-Paul Casanova ◽  
Eric Faure
Keyword(s):  
Physiological Role ◽  
Class Ii ◽  
Class I ◽  
Rrna Genes ◽  
Whole Body ◽  
Rrna Gene ◽  
28S Rrna ◽  
Cellular Functions ◽  
Intraindividual Variation ◽  
Spadella Cephaloptera

AbstractChaetognaths constitute a small marine phylum of approximately 120 species. Two classes of both 18S and 28S rRNA gene sequences have been evidenced in this phylum, even though significant intraindividual variation in the sequences of rRNA genes is unusual in animal genomes. These observations led to the hypothesis that this unusual genetic characteristic could play one or more physiological role(s). Using in situ hybridization on the frontal sections of the chaetognath Spadella cephaloptera, we found that the 18S Class I genes are expressed in the whole body, with a strong expression throughout the gut epithelium, whereas the expression of the 18S Class II genes is restricted to the oocytes. Our results could suggest that the paralog products of the 18S Class I genes are probably the “housekeeping” 18S rRNAs, whereas those of class II would only be essential in specific tissues. These results provide support for the idea that each type of 18S paralog is important for specific cellular functions and is under the control of selective factors.

scholarly journals Testing the higher-level phylogenetic classification of Digenea (Platyhelminthes, Trematoda) based on nuclear rDNA sequences before entering the age of the ‘next-generation’ Tree of Life

2019 ◽  
Vol 93 (3) ◽  
pp. 260-276 ◽  
Author(s):  
G. Pérez-Ponce de León ◽  
D.I. Hernández-Mena
Keyword(s):  
Phylogenetic Relationships ◽  
Phylogenetic Signal ◽  
28S Rdna ◽  
Rrna Genes ◽  
Rrna Gene ◽  
28S Rrna ◽  
Next Generation ◽  
Phylogenetic Classification ◽  
Rdna Sequences

AbstractDigenea Carus, 1863 represent a highly diverse group of parasitic platyhelminths that infect all major vertebrate groups as definitive hosts. Morphology is the cornerstone of digenean systematics, but molecular markers have been instrumental in searching for a stable classification system of the subclass and in establishing more accurate species limits. The first comprehensive molecular phylogenetic tree of Digenea published in 2003 used two nuclear rRNA genes (ssrDNA = 18S rDNA and lsrDNA = 28S rDNA) and was based on 163 taxa representing 77 nominal families, resulting in a widely accepted phylogenetic classification. The genetic library for the 28S rRNA gene has increased steadily over the last 15 years because this marker possesses a strong phylogenetic signal to resolve sister-group relationships among species and to infer phylogenetic relationships at higher levels of the taxonomic hierarchy. Here, we have updated the database of 18S and 28S rRNA genes until December 2017, we have added newly generated 28S rDNA sequences and we have reassessed phylogenetic relationships to test the current higher-level classification of digeneans (at the subordinal and subfamilial levels). The new dataset consisted of 1077 digenean taxa allocated to 106 nominal families for 28S and 419 taxa in 98 families for 18S. Overall, the results were consistent with previous higher-level classification schemes, and most superfamilies and suborders were recovered as monophyletic assemblages. With the advancement of next-generation sequencing (NGS) technologies, new phylogenetic hypotheses from complete mitochondrial genomes have been proposed, although their power to resolve deep levels of trees remains controversial. Since data from NGS methods are replacing other widely used markers for phylogenetic analyses, it is timely to reassess the phylogenetic relationships of digeneans with conventional nuclear rRNA genes, and to use the new analysis to test the performance of genomic information gathered from NGS, e.g. mitogenomes, to infer higher-level relationships of this group of parasitic platyhelminths.

scholarly journals Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

2002 ◽  
Vol 68 (10) ◽  
pp. 5064-5081 ◽  
Author(s):  
Alexander Loy ◽  
Angelika Lehner ◽  
Natuschka Lee ◽  
Justyna Adamczyk ◽  
Harald Meier ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Oligonucleotide Microarray ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Sulfate Reducing ◽  
Specific Pcr

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

The effect of transposon Pokey insertions on sequence variation in the 28S rRNA gene of Daphnia pulex

Genome ◽  
2008 ◽  
Vol 51 (12) ◽  
pp. 988-1000 ◽  
Author(s):  
Shiona K. Glass ◽  
Anna Moszczynska ◽  
Teresa J. Crease
Keyword(s):  
Sequence Variation ◽  
Insertion Site ◽  
Daphnia Pulex ◽  
Rrna Genes ◽  
Rrna Gene ◽  
28S Rrna ◽  
Intraindividual Variation ◽  
Rdna Array ◽  
The Impact ◽  
Host Genes

The goal of this study was to determine the impact of breeding system and the presence of the transposon Pokey on intraindividual variation in 28S rRNA genes. We PCR-amplified, cloned, and sequenced 1000 nucleotides downstream of the Pokey insertion site in genes with and without insertions from 10 obligately and 10 cyclically parthenogenetic isolates of Daphnia pulex. Variation among genes with Pokey insertions was higher than variation among genes without insertions in both cyclic and obligate isolates. Although the differences were not quite significant (p = 0.06 in both cases), the results suggest that Pokey insertions are likely to inhibit the homogenization of their host genes to some extent. We also observed that the complement of 28S rRNA alleles differed between genes with and without inserts in some isolates, suggesting that a particular inserted gene can persist for substantial periods of time and even spread within the rDNA array, despite the fact that insertions are deleterious. This apparently contradictory pattern can be explained if homogenization of rRNA genes occurs primarily by gene conversion, but copies with Pokey inserts can occasionally increase in frequency within arrays owing to unequal crossing over events that do not originate in the inserted genes themselves.

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 Direct and Rapid Detection by PCR ofErysipelothrix sp. DNAs Prepared from Bacterial Strains and Animal Tissues

1999 ◽  
Vol 37 (12) ◽  
pp. 4093-4098 ◽  
Author(s):  
Kouichi Takeshi ◽  
Souichi Makino ◽  
Tetsuya Ikeda ◽  
Noriko Takada ◽  
Atsushi Nakashiro ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gene Cluster ◽  
Dna Sequences ◽  
Screening Method ◽  
Rapid Screening ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Link Type ◽  
Species Specific

A PCR method for rapid screening of Erysipelothrix spp. in the slaughterhouse was carried out by using four species-specific sets of oligonucleotide primers after initial amplification with the primer set MO101-MO102, which amplifies the 16S rRNA sequences of all four Erysipelothrix species. The DNA sequences coding for the rRNA gene cluster, including 16S rRNA, 23S rRNA, and the noncoding region downstream of 5S rRNA, were determined in order to design primers for the species-specific PCR detection system. The homology among the 4.5-kb DNA sequences of the rRNA genes ofErysipelothrix rhusiopathiae serovar 2 (DNA Data Bank of Japan accession no. AB019247), E. tonsillarum serovar 7 (accession no. AB019248), E. rhusiopathiae serovar 13 (accession no. AB019249), and E. rhusiopathiae serovar 18 (accession no. AB019250) ranged from 96.0 to 98.4%. The PCR amplifications were specific and were able to distinguish the DNAs from each of the four Erysipelothrix species. The results of PCR tests performed directly with tissue specimens from diseased animals were compared with the results of cultivation tests, and the PCR tests were completed within 5 h. The test with this species-specific system based on PCR amplification with the DNA sequences coding for the rRNA gene cluster was an accurate, easy-to-read screening method for rapid diagnosis of Erysipelothrix sp. infection in the slaughterhouse.

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