A multiplex real-time PCR assay for the simultaneous quantification of the major plant-parasitic nematodes in Japan

Nematology ◽  
2011 ◽  
Vol 13 (6) ◽  
pp. 713-720 ◽  
Author(s):  
Yu Yu Min ◽  
Keita Goto ◽  
Koki Toyota ◽  
Erika Sato
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Single Species ◽  
The Other ◽  
Parasitic Nematodes ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Pcr Assays

AbstractMultiplex real-time PCR assays were developed to quantify multiple species of Meloidogyne incognita, Pratylenchus penetrans, Globodera rostochiensis and Heterodera glycines in soil. The probes specific for P. penetrans and H. glycines are labelled with a fluorescence molecule, FAM, and those for M. incognita and G. rostochiensis with ROX. The primers and probes are species-specific to P. penetrans, but group-specific to the other species. DNA was extracted from suspensions containing each nematode and multiplex Cycleave® PCR assays were done for pairs of P. penetrans and M. incognita, P. penetrans and G. rostochiensis, or G. rostochiensis and H. glycines. The results revealed that the target nematode, except for H. glycines, was quantified in the presence of less than 100 times that of the other nematode (competitor), but underestimated in the presence of 1000 times the competitor. Such underestimation was solved by the use of SYBR Green I real time PCR assays targeting a single species. Multiplex PCR assay for P. penetrans and M. incognita was done using environmental DNA (eDNA) extracted from a soil naturally infested with the nematodes. Results quantified both species. Multiplex assay using eDNA may enable a sensitive and simultaneous detection of P. penetrans and M. incognita or P. penetrans and G. rostochiensis in soil although caution is needed in case the existing ratio is biased to one of the species.

scholarly journals Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

2010 ◽  
Vol 76 (13) ◽  
pp. 4387-4395 ◽  
Author(s):  
Sebastian Kirchner ◽  
K. Melanie Krämer ◽  
Martin Schulze ◽  
Diana Pauly ◽  
Daniela Jacob ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Target Genes ◽  
False Negative ◽  
Simultaneous Detection ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Pcr Assays

ABSTRACT Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 103 and 105 CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.

scholarly journals Simultaneous detection and differentiation of clinically relevant relapsing fever Borrelia with semi-multiplex real-time PCR

2021 ◽  
Author(s):  
Elizabeth A. Dietrich ◽  
Adam J. Replogle ◽  
Sarah W. Sheldon ◽  
Jeannine M. Petersen
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Hard Tick ◽  
Emerging Pathogens ◽  
Pcr Assay ◽  
Relapsing Fever ◽  
Multiplex Pcr Assay

Bacterial vector-borne diseases, including Borrelia species, present a significant diagnostic, clinical, and public health challenge due to their overlapping symptoms and the breadth of causative agents and arthropod vectors. The relapsing fever (RF) borreliae encompass both established and emerging pathogens and are transmitted to humans by soft ticks, hard ticks, or lice. We developed a real-time semi-multiplex PCR assay that detects multiple RF borreliae causing human illness and classifies them into one of three groups. The groups are based on genetic similarity and include agents of soft-tick relapsing fever (B. hermsii and others), the emerging hard tick transmitted pathogen B. miyamotoi, and the agent of louse-borne relapsing fever (B. recurrentis). The real-time PCR assay uses a single primer pair designed to amplify all known pathogenic RF borreliae, and multiple TaqMan probes to allow for detection of and differentiation among the three groups. The assay detects all RF borreliae tested with an analytical limit of detection below 15 genome equivalents per reaction. Thirty isolates of RF borreliae encompassing six species were accurately identified. Thirty-nine of 41 residual specimens (EDTA whole blood, serum, or plasma) from patients with RF were detected and correctly classified. None of 42 clinical samples from patients with other infections and 46 culture specimens from non-RF bacteria were detected. The development of a single assay real-time PCR approach will help to improve diagnosis of RF by simplifying the selection of tests to aid in clinical management of acutely ill RF patients.

scholarly journals A Real-Time Multiplex PCR Assay for Detection ofElizabethkingiaSpecies and Differentiation betweenElizabethkingia anophelisandE. meningoseptica

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Aubree J. Kelly ◽  
Sandor E. Karpathy ◽  
Christopher A. Gulvik ◽  
Melissa L. Ivey ◽  
Anne M. Whitney ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Real Time ◽  
Nosocomial Infections ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Diagnostic Tools ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
Elizabethkingia Meningoseptica

ABSTRACTNosocomial infections ofElizabethkingiaspecies can have fatal outcomes if not identified and treated properly. The current diagnostic tools available require culture and isolation, which can extend the reporting time and delay treatment. Using comparative genomics, we developed an efficient multiplex real-time PCR for the simultaneous detection of all known species ofElizabethkingia, as well as differentiating the two most commonly reported species,Elizabethkingia anophelisandElizabethkingia meningoseptica.

scholarly journals Simultaneous Detection of Acidovorax avenae subsp. citrulli and Didymella bryoniae in Cucurbit Seedlots Using Magnetic Capture Hybridization and Real-Time Polymerase Chain Reaction

2009 ◽  
Vol 99 (6) ◽  
pp. 666-678 ◽  
Author(s):  
Y. Ha ◽  
A. Fessehaie ◽  
K. S. Ling ◽  
W. P. Wechter ◽  
A. P. Keinath ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection Threshold ◽  
Simultaneous Detection ◽  
Didymella Bryoniae ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Magnetic Capture ◽  
Polymerase Chain ◽  
Pcr Assays

To improve the simultaneous detection of two pathogens in cucurbit seed, a combination of magnetic capture hybridization (MCH) and multiplex real-time polymerase chain reaction (PCR) was developed. Single-stranded DNA hybridization capture probes targeting DNA of Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, and Didymella bryoniae, causal agent of gummy stem blight, were covalently attached to magnetic particles and used to selectively concentrate template DNA from cucurbit seed samples. Sequestered template DNAs were subsequently amplified by multiplex real-time PCR using pathogen-specific TaqMan PCR assays. The MCH multiplex real-time PCR assay displayed a detection threshold of A. avenae subsp. citrulli at 10 CFU/ml and D. bryoniae at 105 conidia/ml in mixtures of pure cultures of the two pathogens, which was 10-fold more sensitive than the direct real-time PCR assays for the two pathogens separately. Although the direct real-time PCR assay displayed a detection threshold for A. avenae subsp. citrulli DNA of 100 fg/μl in 25% (1/4 samples) of the samples assayed, MCH real-time PCR demonstrated 100% detection frequency (4/4 samples) at the same DNA concentration. MCH did not improve detection sensitivity for D. bryoniae relative to direct real-time PCR using conidial suspensions or seed washes from D. bryoniae-infested cucurbit seed. However, MCH real-time PCR facilitated detection of both target pathogens in watermelon and melon seed samples (n = 5,000 seeds/sample) in which 0.02% of the seed were infested with A. avenae subsp. citrulli and 0.02% were infested with D. bryoniae.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals Inter-laboratory comparison of three different real-time PCR assays for the detection of Pneumocystis jiroveci in bronchoalveolar lavage fluid samples

2006 ◽  
Vol 55 (9) ◽  
pp. 1229-1235 ◽  
Author(s):  
Catharina F. M. Linssen ◽  
Jan A. Jacobs ◽  
Pieter Beckers ◽  
Kate E. Templeton ◽  
Judith Bakkers ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Real Time ◽  
Real Time Pcr ◽  
Bronchoalveolar Lavage Fluid ◽  
Medical Center ◽  
Tertiary Care ◽  
Lavage Fluid ◽  
The Other ◽  
Pneumocystis Jiroveci ◽  
Pcr Assays

Pneumocystis jiroveci pneumonia (PCP) is an opportunistic infection affecting immunocompromised patients. While conventional diagnosis of PCP by microscopy is cumbersome, the use of PCR to diagnose PCP has great potential. Nevertheless, inter-laboratory validation and standardization of PCR assays is lacking. The aim of this study was to evaluate the inter-laboratory agreement of three independently developed real-time PCR assays for the detection of P. jiroveci in bronchoalveolar lavage fluid samples. Therefore, 124 samples were collected in three tertiary care laboratories (Leiden University Medical Center, Maastricht Infection Center and Radboud University Nijmegen Medical Centre) and were tested by both microscopy and real-time PCR. Of 41 samples positive for P. jiroveci by microscopy, 40 were positive in all three PCR assays. The remaining sample was positive in a single assay only. Out of 83 microscopy-negative samples, 69 were negative in all three PCR assays. The other 14 samples were found positive, either in all three assays (n=5), in two (n=2) or in one of the assays (n=7). The data demonstrate high inter-laboratory agreement among real-time PCR assays for the detection of P. jiroveci.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

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