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 Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

2004 ◽  
Vol 70 (5) ◽  
pp. 3171-3175 ◽  
Author(s):  
X. Bonjoch ◽  
E. Ballesté ◽  
A. R. Blanch
Keyword(s):  
16S Rrna ◽  
Multiplex Pcr ◽  
Fecal Pollution ◽  
Sensitivity Threshold ◽  
Rrna Gene ◽  
Specific Primers ◽  
Animal Wastewater ◽  
Wastewater Samples ◽  
Species Specific ◽  
Different Origins

ABSTRACT Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

scholarly journals Detection of Pythium ultimum Using Polymerase Chain Reaction with Species-Specific Primers

Plant Disease ◽  
1997 ◽  
Vol 81 (10) ◽  
pp. 1155-1160 ◽  
Author(s):  
K. Kageyama ◽  
A. Ohyama ◽  
M. Hyakumachi
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Transcribed Spacer ◽  
Pcr Amplification ◽  
Its Region ◽  
Pythium Ultimum ◽  
Pythium Species ◽  
Chain Reaction ◽  
Specific Primers ◽  
Polymerase Chain ◽  
Species Specific

This study was conducted to sequence the rDNA internal transcribed spacer (ITS) region of Pythium ultimum and Pythium group HS, design species-specific primers for polymerase chain reaction (PCR), and detect P. ultimum from diseased seedlings using PCR. The sequence of the ITS region of P. ultimum was identical with that of Pythium group HS. The results support the reports that the HS group is an asexual strain of P. ultimum. Using PCR, the primer pair K1+K3, designed on portions of the sequence of the ITS region, amplified isolates of P. ultimum and the HS group but not isolates of 20 other Pythium species. DNA extracts from damped-off seedlings were not amplified, but a 10-fold dilution of the extracts with Tris-EDTA (TE) buffer diluted the inhibitors and allowed PCR amplification. The primer pair used detected P. ultimum from a single diseased seedling.

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.

Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies

2021 ◽  
pp. 104063872110634
Author(s):  
Barbara Ujvári ◽  
Hubert Gantelet ◽  
Tibor Magyar
Keyword(s):  
Multiplex Pcr ◽  
Pasteurella Multocida ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Multiplex Pcr Assay ◽  
Subspecies Level ◽  
Species Specific ◽  
Reference Strains

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

scholarly journals Biotyping and Antimicrobial Susceptibility of Enterococcus faecalis and E. faecium Isolated from Urine and Stool Samples

2021 ◽  
Vol 13 (10) ◽  
Author(s):  
Gülşah Tollu ◽  
Ismail Hakkı Ekin
Keyword(s):  
Multiplex Pcr ◽  
Antimicrobial Susceptibility ◽  
Animal Health ◽  
Molecular Techniques ◽  
Penicillin G ◽  
Clinical Approach ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Stool Samples ◽  
Species Specific

Background: Enterococci are one of the opportunistic pathogenic microorganisms that can cause significant problems for human and animal health. Enterococcus faecium seems to be more resistant to antibiotics than E. faecalis. It is thought that pathogenic E. faecium can develop antibiotic resistance very quickly, and the ability to transfer this feature is considered to be an important health risk. Objectives: This study aimed to determine the prevalence, biotypes, and in vitro antimicrobial susceptibility of E. faecalis and E. faecium strains isolated from 267 routine urine and stool samples that were brought to the microbiology laboratory of Regional Training and Research Hospital of Van, with permission of the patients. Methods: In the present study, enterococci using species-specific primers to examine E. faecalis and E. faecium multiplex PCR technique was applied. Biotyping of the isolates was used to identify them as E. faecalis and E. faecium by molecular techniques, and antibiotic susceptibility of all samples was examined, as well. Results: The isolates were identified by multiplex PCR using species-specific primers for E. faecalis and E. faecium. Biotyping based on 13 biochemical tests showed that 72.5%, 12.5%, and 15% of E. faecalis strains were of biotypes I, II, and III, respectively, whereas E. faecium strains could be divided into biotype I (10%), biotype II (12.5%), biotype III (27.5%), and biotype IV (50%). Additionally, all E. faecalis strains were found to be susceptible to penicillin G and imipenem. On the other hand, 95% of the E. faecalis strains were found to be resistant to clindamycin, 77.5% to tetracycline and trimethoprim/sulfamethoxazole, 42.5% to erythromycin, 32.5% to gentamicin, and 17.5% to ciprofloxacin. Of E. faecium strains, 37.5% were found to be resistant to clindamycin, 32.5% to penicillin G, 27.5% to erythromycin and imipenem, 20% to ciprofloxacin, 17.5% to tetracycline and trimethoprim/sulfamethoxazole, 15% to gentamicin, and 5% to vancomycin. Conclusions: In conclusion, the identification of E. faecalis and E. faecium strains by PCR is reliable and faster than biochemical tests. Additionally, the results of antimicrobial susceptibility tests may provide important contributions to the clinical approach.

Rapid identification of Saccharomonospora strains by multiplex PCR using species-specific primers within the 16S rRNA gene

1996 ◽  
Vol 27 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Jung-Hoon Yoon ◽  
Sung Taik Lee ◽  
Yong Kook Shin ◽  
Sam-Bong Kim ◽  
Hong-Joong Kim ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Multiplex Pcr ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Specific Primers ◽  
Species Specific ◽  
The 16S Rrna Gene
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