scholarly journals SpeciesPrimer: a bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8544
Author(s):  
Matthias Dreier ◽  
Hélène Berthoud ◽  
Noam Shani ◽  
Daniel Wechsler ◽  
Pilar Junier
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Bacterial Species ◽  
Primer Design ◽  
Diagnostic Methods ◽  
Food Microbiology ◽  
Fermented Foods ◽  
Bioinformatics Pipeline ◽  
Specific Primers ◽  
Species Specific

Background Quantitative real-time PCR (qPCR) is a well-established method for detecting and quantifying bacteria, and it is progressively replacing culture-based diagnostic methods in food microbiology. High-throughput qPCR using microfluidics brings further advantages by providing faster results, decreasing the costs per sample and reducing errors due to automatic distribution of samples and reagents. In order to develop a high-throughput qPCR approach for the rapid and cost-efficient quantification of microbial species in complex systems such as fermented foods (for instance, cheese), the preliminary setup of qPCR assays working efficiently under identical PCR conditions is required. Identification of target-specific nucleotide sequences and design of specific primers are the most challenging steps in this process. To date, most available tools for primer design require either laborious manual manipulation or high-performance computing systems. Results We developed the SpeciesPrimer pipeline for automated high-throughput screening of species-specific target regions and the design of dedicated primers. Using SpeciesPrimer, specific primers were designed for four bacterial species of importance in cheese quality control, namely Enterococcus faecium, Enterococcus faecalis, Pediococcus acidilactici and Pediococcus pentosaceus. Selected primers were first evaluated in silico and subsequently in vitro using DNA from pure cultures of a variety of strains found in dairy products. Specific qPCR assays were developed and validated, satisfying the criteria of inclusivity, exclusivity and amplification efficiencies. Conclusion In this work, we present the SpeciesPrimer pipeline, a tool to design species-specific primers for the detection and quantification of bacterial species. We use SpeciesPrimer to design qPCR assays for four bacterial species and describe a workflow to evaluate the designed primers. SpeciesPrimer facilitates efficient primer design for species-specific quantification, paving the way for a fast and accurate quantitative investigation of microbial communities.

scholarly journals SpeciesPrimer: A bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species

2019 ◽  
Author(s):  
Matthias Dreier ◽  
Hélène Berthoud ◽  
Noam Shani ◽  
Daniel Wechsler ◽  
Pilar Junier
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Bacterial Species ◽  
Primer Design ◽  
Diagnostic Methods ◽  
Food Microbiology ◽  
Bioinformatics Pipeline ◽  
Specific Primers ◽  
Species Specific

Background. Quantitative real-time PCR (qPCR) is a well-established method for detecting and quantifying bacteria, and it is progressively replacing culture-based diagnostic methods in food microbiology. High-throughput qPCR using microfluidics brings further advantages by providing faster results, decreasing the costs per sample and reducing errors due to automatic distribution of samples and reactants. In order to develop a high-throughput qPCR approach for the rapid and cost-efficient quantification of microbial species in a given system (for instance, cheese), the preliminary setup of qPCR assays working efficiently under identical PCR conditions is required. Identification of target-specific nucleotide sequences and design of specific primers are the most challenging steps in this process. To date, most available tools for primer design require either laborious manual manipulation or high-performance computing systems. Results. We developed the SpeciesPrimer pipeline for automated high-throughput screening of species-specific target regions and the design of dedicated primers. Using SpeciesPrimer specific primers were designed for four bacterial species of importance in cheese quality control, namely Enterococcus faecium, Enterococcus faecalis, Pediococcus acidilactici and Pediococcus pentosaceus. Selected primers were first evaluated in silico and subsequently in vitro using DNA from pure cultures of a variety of strains found in dairy products. Specific qPCR assays were developed and validated, satisfying the criteria of inclusivity, exclusivity and amplification efficiencies. Conclusion. In this work, we present the SpeciesPrimer pipeline, a tool to design species-specific primers for the detection and quantification of bacterial species. We use SpeciesPrimer to design qPCR assays for four bacterial species and describe a workflow to evaluate the designed primers. SpeciesPrimer facilitates efficient primer design for species-specific quantification, paving the way for a fast and accurate quantitative investigation of microbial communities.

scholarly journals SpeciesPrimer: A bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species

2019 ◽  
Author(s):  
Matthias Dreier ◽  
Hélène Berthoud ◽  
Noam Shani ◽  
Daniel Wechsler ◽  
Pilar Junier
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Bacterial Species ◽  
Primer Design ◽  
Diagnostic Methods ◽  
Food Microbiology ◽  
Bioinformatics Pipeline ◽  
Specific Primers ◽  
Species Specific

Background. Quantitative real-time PCR (qPCR) is a well-established method for detecting and quantifying bacteria, and it is progressively replacing culture-based diagnostic methods in food microbiology. High-throughput qPCR using microfluidics brings further advantages by providing faster results, decreasing the costs per sample and reducing errors due to automatic distribution of samples and reactants. In order to develop a high-throughput qPCR approach for the rapid and cost-efficient quantification of microbial species in a given system (for instance, cheese), the preliminary setup of qPCR assays working efficiently under identical PCR conditions is required. Identification of target-specific nucleotide sequences and design of specific primers are the most challenging steps in this process. To date, most available tools for primer design require either laborious manual manipulation or high-performance computing systems. Results. We developed the SpeciesPrimer pipeline for automated high-throughput screening of species-specific target regions and the design of dedicated primers. Using SpeciesPrimer specific primers were designed for four bacterial species of importance in cheese quality control, namely Enterococcus faecium, Enterococcus faecalis, Pediococcus acidilactici and Pediococcus pentosaceus. Selected primers were first evaluated in silico and subsequently in vitro using DNA from pure cultures of a variety of strains found in dairy products. Specific qPCR assays were developed and validated, satisfying the criteria of inclusivity, exclusivity and amplification efficiencies. Conclusion. In this work, we present the SpeciesPrimer pipeline, a tool to design species-specific primers for the detection and quantification of bacterial species. We use SpeciesPrimer to design qPCR assays for four bacterial species and describe a workflow to evaluate the designed primers. SpeciesPrimer facilitates efficient primer design for species-specific quantification, paving the way for a fast and accurate quantitative investigation of microbial communities.

scholarly journals Development of Primers and Probes for Genus and Species Specific Detection of ‘Candidatus Liberibacter Species’ by Real-Time PCR

Plant Disease ◽  
2013 ◽  
Vol 97 (9) ◽  
pp. 1235-1243 ◽  
Author(s):  
G. Ananthakrishnan ◽  
N. Choudhary ◽  
Avijit Roy ◽  
V. G. Sengoda ◽  
E. Postnikova ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Rrna Gene ◽  
Single Pair ◽  
Degenerate Primers ◽  
Specific Primers ◽  
Genus Level ◽  
Candidatus Liberibacter ◽  
Species Specific

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different ‘Candidatus Liberibacter species’, ‘Ca. Liberibacter asiaticus’, ‘Ca. Liberibacter americanus’, and ‘Ca. Liberibacter africanus’, which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of ‘Ca. Liberibacter’ at the genus level, providing increased convenience. Recent molecular analyses of ‘Ca. Liberibacter species’ and other bacteria suggest that the rpoB gene (encoding the β-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus ‘Ca. Liberibacter species’, enabling detection of ‘Ca. Liberibacter’ at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected ‘Ca. Liberibacter solanacearum’, a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

Agronomically important thrips: development of species-specific primers in multiplex PCR and microarray assay using internal transcribed spacer 1 (ITS1) sequences for identification

2014 ◽  
Vol 105 (1) ◽  
pp. 52-59 ◽  
Author(s):  
W.B. Yeh ◽  
M.J. Tseng ◽  
N.T. Chang ◽  
S.Y. Wu ◽  
Y.S. Tsai
Keyword(s):  
Multiplex Pcr ◽  
Species Identification ◽  
Internal Transcribed Spacer ◽  
High Throughput Screening ◽  
Virus Transmission ◽  
Specific Primers ◽  
Its1 Sequence ◽  
Microarray Assay ◽  
Thrips Species ◽  
Species Specific

AbstractThrips, the sole vector of plantTospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.

scholarly journals New Species-Specific Primers for Molecular Diagnosis of Bactrocera minax and Bactrocera tsuneonis (Diptera: Tephritidae) in China Based on DNA Barcodes

Insects ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 447 ◽  
Author(s):  
Linyu Zheng ◽  
Yue Zhang ◽  
Wenzhao Yang ◽  
Yiying Zeng ◽  
Fan Jiang ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Dna Barcode ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Specific Primers ◽  
Oxidase Subunit ◽  
Preimaginal Stages ◽  
Specific Fragment ◽  
Species Specific

Tephritidae fruit flies (Diptera: Tephritidae) are regarded as important damage-causing species due to their ability to cause great economic losses in fruit and vegetable crops. Bactrocera minax and Bactrocera tsuneonis are two sibling species of the subgenus Tetradacus of Bactrocera that are distributed across a limited area of China, but have caused serious impacts. They share similar morphological characteristics. These characteristics can only be observed in the female adult individuals. The differences between them cannot be observed in preimaginal stages. Thus, it is difficult to distinguish them in preimaginal stages morphologically. In this study, we used molecular diagnostic methods based on cytochrome c oxidase subunit I and species-specific markers to identify these two species and improve upon the false-positive results of previous species-detection primers. DNA barcode sequences were obtained from 900 individuals of B. minax and 63 individuals of B. tsuneonis. Based on these 658 bp DNA barcode sequences of the cytochrome c oxidase subunit I gene, we successfully designed the species-specific primers for B. minax and B. tsuneonis. The size of the B. minax specific fragment was 422 bp and the size of the B. tsuneonis specific fragment was 456 bp. A series of PCR trials ensured the specificity of these two pairs of primers. Sensitivity assay results demonstrated that the detection limit for the DNA template concentration was 0.1~1 ng/μL for these two species. In this study, we established a more reliable, rapid, and low-cost molecular identification method for all life stages of B. minax and B. tsuneonis. Species-specific PCR can be applied in plant quarantine, monitoring and control of B. minax and B. tsuneonis.

scholarly journals Development of a High-Throughput Microfluidic qPCR System for the Quantitative Determination of Quality-Relevant Bacteria in Cheese

2021 ◽  
Vol 11 ◽  
Author(s):  
Matthias Dreier ◽  
Hélène Berthoud ◽  
Noam Shani ◽  
Daniel Wechsler ◽  
Pilar Junier
Keyword(s):  
High Throughput ◽  
Bacterial Species ◽  
Microbial Community Composition ◽  
Fermented Foods ◽  
Polymerase Chain ◽  
Cost Efficient ◽  
The Cost ◽  
Opening Up ◽  
Mock Communities

The composition of the cheese microbiome has an important impact on the sensorial quality and safety of cheese. Therefore, much effort has been made to investigate the microbial community composition of cheese. Quantitative real-time polymerase chain reaction (qPCR) is a well-established method for detecting and quantifying bacteria. High-throughput qPCR (HT-qPCR) using microfluidics brings further advantages by providing fast results and by decreasing the cost per sample. We have developed a HT-qPCR approach for the rapid and cost-efficient quantification of microbial species in cheese by designing qPCR assays targeting 24 species/subspecies commonly found in cheese. Primer pairs were evaluated on the Biomark (Fluidigm) microfluidic HT-qPCR system using DNA from single strains and from artificial mock communities. The qPCR assays worked efficiently under identical PCR conditions, and the validation showed satisfying inclusivity, exclusivity, and amplification efficiencies. Preliminary results obtained from the HT-qPCR analysis of DNA samples of model cheeses made with the addition of adjunct cultures confirmed the potential of the microfluidic HT-qPCR system to screen for selected bacterial species in the cheese microbiome. HT-qPCR data of DNA samples of two downgraded commercial cheeses showed that this approach provides valuable information that can help to identify the microbial origin of quality defects. This newly developed HT-qPCR system is a promising approach that will allow simultaneous monitoring of quality-relevant species in fermented foods with high bacterial diversity, thereby opening up new perspectives for the control and assurance of high product quality.

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 PrimerServer: a high-throughput primer design and specificity-checking platform

2017 ◽  
Author(s):  
Tao Zhu ◽  
Chengzhen Liang ◽  
Zhigang Meng ◽  
Yanyan Li ◽  
Yayu Wu ◽  
...  
Keyword(s):  
High Throughput ◽  
Molecular Breeding ◽  
Primer Design ◽  
Command Line ◽  
Specific Primers ◽  
Data Presentation ◽  
Medical Testing ◽  
Link Type ◽  
Fast Running

AbstractSummaryDesigning specific primers for multiple sites across the whole genome is still challenging, especially in species with complex genomes. Here we present PrimerServer, a high-throughput primer design and specificity-checking platform with both web and command-line interfaces. This platform efficiently integrates site selection, primer design, specificity checking and data presentation. In our case study, PrimerServer achieved high accuracy and a fast running speed for a large number of sites, suggesting its potential for molecular biology applications such as molecular breeding or medical testing.Availability and ImplementationSource code for PrimerServer is available at https://github.com/billzt/PrimerServer. A demo server is freely accessible at https://primerserver.org, with all major browsers [email protected] or [email protected]

scholarly journals Two-Step PCR-Based Assay for Identification of Bacterial Etiology of Otitis Media with Effusion in Infected Lebanese Children

1998 ◽  
Vol 36 (5) ◽  
pp. 1185-1188 ◽  
Author(s):  
Ghassan M. Matar ◽  
Nada Sidani ◽  
Michel Fayad ◽  
Usamah Hadi
Keyword(s):  
Otitis Media ◽  
Otitis Media With Effusion ◽  
Bacterial Species ◽  
Cost Effective ◽  
Universal Primers ◽  
Rrna Gene ◽  
Specific Primer ◽  
Specific Primers ◽  
Bacterial Dna ◽  
Species Specific

We developed and evaluated a two-step PCR-based assay with universal primers and genus- or species-specific primers for the detection of the most prevalent bacterial etiologies of otitis media with effusion (OME) in children from Lebanese hospitals. These etiologies included Haemophilus, Streptococcus, and Moraxella (Branhamella)catarrhalis, which were detected in middle-ear effusion (MEE) samples taken from children with OME. A total of 47 MEE samples were aspirated from 36 patients during insertion of a tympanostomy tube performed particularly for OME. The duration of effusion in all patients was ≥2 months. DNA was extracted from MEE samples, and PCR was initially done with DNA extracts by using the universal primers RW01 and DG74, which flank an ∼370-bp fragment found in the 16S rRNA gene of all bacterial species. For the identification of specific bacteria, we used in three separate reaction mixtures the following genus- or species-specific primers: (i) aHaemophilus-specific probe (probe RDR125) as a primer along with DG74, (ii) a Streptococcus-specific primer (primer STR1; designed by us) along with DG74, and (iii) an M. catarrhalis-specific primer pair (primer pair MCA1-MCA2). Thirty-five MEE samples (74.5%) gave the expected 370-bp band, indicating the presence of bacterial DNA in the tested samples. Of the 35 PCR-positive samples tested, 33 (94.3%) were positive forHaemophilus, 3 (8.6%) were positive forStreptococcus, and 10 (28.6%) were positive for M. catarrhalis. Ten samples (28.6%) exhibited a mixed infection and were positive for both Haemophilus and M. catarrhalis. Culture was simultaneously performed for all 47 MEE samples. Ten of the 47 MEE samples (21.3%) exhibited bacterial growth. These 10 were PCR positive for bacterial DNA. The remaining 25 PCR-positive samples were negative by culture, thus showing about 53% discordance between PCR results and those of culture. The PCR assay proved to be more sensitive than culture, more rapid, less cumbersome, and more cost-effective than the available PCR-Southern hybridization-based assays.

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