scholarly journals PhyloPrimer: a taxon-specific oligonucleotide design platform

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11120
Author(s):  
Gilda Varliero ◽  
Jared Wray ◽  
Cédric Malandain ◽  
Gary Barker
Keyword(s):  
Microbial Communities ◽  
Nearest Neighbor ◽  
Pcr Primers ◽  
Main Function ◽  
Gene Sequences ◽  
Specific Primers ◽  
Dynamic Tree ◽  
Or Gene ◽  
Species Specific ◽  
Mock Communities

Many environmental and biomedical biomonitoring and detection studies aim to explore the presence of specific organisms or gene functionalities in microbiome samples. In such cases, when the study hypotheses can be answered with the exploration of a small number of genes, a targeted PCR-approach is appropriate. However, due to the complexity of environmental microbial communities, the design of specific primers is challenging and can lead to non-specific results. We designed PhyloPrimer, the first user-friendly platform to semi-automate the design of taxon-specific oligos (i.e., PCR primers) for a gene of interest. The main strength of PhyloPrimer is the ability to retrieve and align GenBank gene sequences matching the user’s input, and to explore their relationships through an online dynamic tree. PhyloPrimer then designs oligos specific to the gene sequences selected from the tree and uses the tree non-selected sequences to look for and maximize oligo differences between targeted and non-targeted sequences, therefore increasing oligo taxon-specificity (positive/negative consensus approach). Designed oligos are then checked for the presence of secondary structure with the nearest-neighbor (NN) calculation and the presence of off-target matches with in silico PCR tests, also processing oligos with degenerate bases. Whilst the main function of PhyloPrimer is the design of taxon-specific oligos (down to the species level), the software can also be used for designing oligos to target a gene without any taxonomic specificity, for designing oligos from preselected sequences and for checking predesigned oligos. We validated the pipeline on four commercially available microbial mock communities using PhyloPrimer to design genus- and species-specific primers for the detection of Streptococcus species in the mock communities. The software performed well on these mock microbial communities and can be found at https://www.cerealsdb.uk.net/cerealgenomics/phyloprimer.

Molecular markers for Peristenus spp. (Hymenoptera: Braconidae) parasitoids associated with Lygus spp. (Hemiptera: Miridae)

2003 ◽  
Vol 135 (1) ◽  
pp. 71-83 ◽  
Author(s):  
M. Erlandson ◽  
L. Braun ◽  
D. Baldwin ◽  
J. Soroka ◽  
M. Ashfaq ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Dna Sequence ◽  
Sequence Data ◽  
Pcr Primers ◽  
Species Level ◽  
Rrna Genes ◽  
Specific Primers ◽  
Canadian Prairies ◽  
Dna Sequence Data ◽  
Species Specific

AbstractMolecular markers for identifying Peristenus spp. parasitoids to species level and preliminary molecular markers to distinguish two groups of Lygus spp. common to the Canadian prairies were developed. Peristenus species-specific polymerase chain reaction (PCR) primers were developed based on DNA sequence data from a 1600-bp region of the internal transcribed spacer region between the 5.8S and 18S nuclear rRNA genes (ITS2). These primers were able to distinguish Peristenus digoneutis Loan, Peristenus stygicus Loan, and Peristenus pallipes (Curtis). Their ability to identify to species-level parasites dissected from field-collected Lygus spp. nymphs was examined by analysis of DNA from 100 parasite samples. Of those samples showing positive PCR amplification with both control (ITS2) and species-specific primers, all were positive for P. pallipes; none of the samples amplified appropriately sized products with P. digoneutis specific or P. stygicus specific primers. These findings were validated using restriction enzyme digests of amplified regions of the Peristenus spp. cytochrome oxidase 1 gene. Both methods were consistent with earlier studies that showed P. pallipes to be the only species of the genus Peristenus to be associated with Lygus spp. on the Canadian prairies. PCR primers based on DNA sequence data from a 550-bp region of the mitochondrial 16S rRNA gene were designed to discriminate Lyguslineolaris (Palisot de Beauvois) from Lygus borealis (Kelton), and Lygus elisus (Van Duzee). These PCR primers were used to identify field-collected nymphs, with most being identified as either L. borealis/L. elisus (72–82%) orL. lineolaris (14–18%). These estimates of species composition closely reflected those of subsequent adult population surveys from the same fields.

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 Detection and Identification of Mycobacteria by Amplification of the Internal Transcribed Spacer Regions with Genus- and Species-Specific PCR Primers

2000 ◽  
Vol 38 (11) ◽  
pp. 4080-4085 ◽  
Author(s):  
Heekyung Park ◽  
Hyunjung Jang ◽  
Cheolmin Kim ◽  
Byungseon Chung ◽  
Chulhun L. Chang ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Bacterial Species ◽  
Its Region ◽  
Pcr Primers ◽  
Culture Collection ◽  
Clinical Isolates ◽  
Mycobacterial Species ◽  
Specific Primers ◽  
Specific Pcr ◽  
Species Specific

We evaluated the usefulness of PCR assays that target the internal transcribed spacer (ITS) region for identifying mycobacteria at the species level. The conservative and species-specific ITS sequences of 33 species of mycobacteria were analyzed in a multialignment analysis. One pair of panmycobacterial primers and seven pairs of mycobacterial species-specific primers were designed. All PCRs were performed under the same conditions. The specificities of the primers were tested with type strains of 20 mycobacterial species from the American Type Culture Collection; 205 clinical isolates of mycobacteria, including 118Mycobacterium tuberculosis isolates and 87 isolates of nontuberculous mycobacteria from 10 species; and 76 clinical isolates of 28 nonmycobacterial pathogenic bacterial species. PCR with the panmycobacterial primers amplified fragments of approximately 270 to 400 bp in all mycobacteria. PCR with the M. tuberculosiscomplex-specific primers amplified an approximately 120-bp fragment only for the M. tuberculosis complex. Multiplex PCR with the panmycobacterial primers and the M. tuberculosiscomplex-specific primers amplified two fragments that were specific for all mycobacteria and the M. tuberculosis complex, respectively. PCR with M. avium complex-, M. fortuitum-, M. chelonae-, M. gordonae-, M. scrofulaceum-, andM. szulgai-specific primers amplified specific fragments only for the respective target organisms. These novel primers can be used to detect and identify mycobacteria simultaneously under the same PCR conditions. Furthermore, this protocol facilitates early and accurate diagnosis of mycobacteriosis.

scholarly journals Species-specific RT-PCR amplification of human enteroviruses: a tool for rapid species identification of uncharacterized enteroviruses

2006 ◽  
Vol 87 (1) ◽  
pp. 119-128 ◽  
Author(s):  
M. Steven Oberste ◽  
Kaija Maher ◽  
Alford J. Williams ◽  
Naomi Dybdahl-Sissoko ◽  
Betty A. Brown ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Pcr Primers ◽  
Human Enterovirus ◽  
Rt Pcr ◽  
Specific Primer ◽  
Specific Primers ◽  
Typical Application ◽  
Partial Sequencing ◽  
Specific Pcr ◽  
Species Specific

The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 3′-non-translated region of enteroviruses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68·3 % of isolates, while the HEV-A and HEV-C primers accounted for 9·7 and 11·3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6·5 %) were amplified by more than one primer set and eight isolates (4·3 %) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 3′-non-translated region sequences.

scholarly journals Microplate Subtractive Hybridization To Enrich for Bacteroidales Genetic Markers for Fecal Source Identification

2005 ◽  
Vol 71 (6) ◽  
pp. 3179-3183 ◽  
Author(s):  
Linda K. Dick ◽  
Michael T. Simonich ◽  
Katharine G. Field
Keyword(s):  
Water Resource Management ◽  
Management Strategies ◽  
Human Health Risk ◽  
Subtractive Hybridization ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Specific Primers ◽  
New Host ◽  
One Step ◽  
Or Gene

ABSTRACT The ability to identify sources of fecal pollution plays a key role in the analysis of human health risk and the implementation of water resource management strategies. One approach to this problem involves the identification of bacterial lineages or gene sequences that are found exclusively in a particular host species or group. We used subtractive hybridization to enrich for target host-specific fecal Bacteroidales rRNA gene fragments that were different from those of very closely related reference (subtracter) host sources. Target host rRNA gene fragments were hybridized to subtracter rRNA gene fragments immobilized in a microplate well, and target sequences that did not hybridize were cloned and sequenced for PCR primer design. The use of microplates for DNA immobilization resulted in a one-step subtractive hybridization in which the products could be directly amplified with PCR. The new host-specific primers designed from subtracted target fragments differentiated among very closely related Bacteroidales rRNA gene sequences and distinguished between similar fecal sources, such as elk and cow or human and domestic pet (dog).

scholarly journals Phylogenetic analysis of Helicobacter species based on partial gyrB gene sequences

2007 ◽  
Vol 57 (3) ◽  
pp. 444-449 ◽  
Author(s):  
Minna Hannula ◽  
Marja-Liisa Hänninen
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Primers ◽  
Phylogenetic Study ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Specific Pcr ◽  
Species Specific ◽  
The 16S Rrna Gene

Analysis of 16S rRNA gene sequences is one of the most common methods for investigating the phylogeny and taxonomy of bacteria. However, several studies have indicated that the 16S rRNA gene does not distinguish between certain Helicobacter species. We therefore selected for phylogenetic analysis an alternative marker, gyrB, encoding gyrase subunit B. The aim of this investigation was to examine the applicability of gyrB gene fragments (~1100 bp) for the phylogenetic study of 16 Helicobacter species and a total of 33 Helicobacter strains included in this study. Based on the sequenced fragments, a phylogenetic tree was obtained that contained two distinct clusters, with gastric species forming one cluster and enterohepatic species the other. The only exception was the gastric species Helicobacter mustelae, which clustered with the enterohepatic species. The calculated similarity matrix revealed the highest interspecies similarity between Helicobacter salomonis and Helicobacter felis (89 %) and the lowest similarity between Helicobacter pullorum and H. felis (60 %). The DNA G+C content of the sequenced fragments was ⩽40 mol% in enterohepatic species and >46 mol% in gastric species, excluding Helicobacter pylori and H. mustelae, with G+C contents of 34 and 42 mol%, respectively. In summary, the gyrB gene fragments provided superior resolution and reliability to the 16S rRNA gene for differentiating between closely related Helicobacter species. A further outcome of this study was achieved by designing gyrB gene-based species-specific PCR primers for the identification of Helicobacter bizzozeronii.

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