scholarly journals Quantification of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains in the Plant Rhizosphere by Real-Time PCR

2007 ◽  
Vol 73 (17) ◽  
pp. 5531-5538 ◽  
Author(s):  
Olga V. Mavrodi ◽  
Dmitri V. Mavrodi ◽  
Linda S. Thomashow ◽  
David M. Weller
Keyword(s):  
Pseudomonas Fluorescens ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Plant Root ◽  
Population Densities ◽  
Pcr Assay ◽  
Wheat Roots ◽  
Specific Pcr ◽  
Pure Cultures

ABSTRACT A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD +) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD + P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (CT s) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD + strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD + pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r 2 = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

Multiplex Real-Time PCR Detection of Fumonisin-Producing and Trichothecene-Producing Groups of Fusarium Species

2004 ◽  
Vol 67 (3) ◽  
pp. 536-543 ◽  
Author(s):  
B. H. BLUHM ◽  
M. A. COUSIN ◽  
C. P. WOLOSHUK
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Fusarium Species ◽  
Fusarium Spp ◽  
Pcr Assay ◽  
Traditional Methods ◽  
Microbiological Methods ◽  
Pcr Products ◽  
Its Pcr

Some species of Fusarium can produce mycotoxins during food processing procedures that facilitate fungal growth, such as the malting of barley. The objectives of this study were to develop a 5′ fluorogenic (Taqman) real-time PCR assay for group-specific detection of trichothecene- and fumonisin-producing Fusarium spp. and to identify Fusarium graminearum and Fusarium verticillioides in field-collected barley and corn samples. Primers and probes were designed from genes involved in mycotoxin biosynthesis (TRI6 and FUM1), and for a genus-specific internal positive control, primers and a probe were designed from Fusarium rDNA sequences. Real-time PCR conditions were optimized for amplification of the three products in a single reaction format. The specificity of the assay was confirmed by testing 9 Fusarium spp. and 33 non- Fusarium fungal species. With serial dilutions of purified genomic DNA from F. verticillioides, F. graminearum, or both as the template, the detection limit of the assay was 5 pg of genomic DNA per reaction. The three products were detectable over four orders of magnitude of template concentration (5 pg to 5 ng of genomic DNA per reaction); at 50 ng template per reaction, only the TRI6 and FUM1 PCR products were detected. Barley and corn samples were evaluated for the presence of Fusarium spp. with traditional microbiological methods and with the real-time PCR assay. The 20 barley samples and 1 corn sample that contained F. graminearum by traditional methods of analysis tested positive for the TRI6 and internal transcribedspacer (ITS) PCR products. The five corn samples that tested positive for F. verticillioides by traditional methods also were positive for the FUM1 and ITS PCR products. These results indicate that the described multiplex real-time PCR assay provides sensitive and accurate differential detection of fumonisin- and trichothecene-producing groups of Fusarium spp. in complex matrices.

scholarly journals Quantitative Molecular Detection of Xanthomonas hortorum pv. carotae in Carrot Seed Before and After Hot-Water Treatment

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1585-1592 ◽  
Author(s):  
Todd N. Temple ◽  
Lindsey J. du Toit ◽  
Michael L. Derie ◽  
Kenneth B. Johnson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Hot Water ◽  
Pcr Assay ◽  
The Real ◽  
Molecular Assays ◽  
Treated Seed ◽  
Carrot Seed ◽  
Pure Cultures

Molecular assays to detect and quantify DNA from viable cells of the seedborne pathogen Xanthomonas hortorum pv. carotae in carrot seed were developed and evaluated for use on nontreated and hot-water-treated seed lots. Both a TaqMan real-time polymerase chain reaction (PCR) assay and a loop-mediated isothermal amplification (LAMP) dilution endpoint assay detected and quantified DNA from viable pathogen cells after treatment of carrot seed washes with the live-dead discriminating dye propidium monoazide (PMA). The detection limits of the assays were approximately 101 CFU for pure cultures of X. hortorum pv. carotae, and 102 to 103 CFU/g seed from naturally infested carrot seed lots. X. hortorum pv. carotae in and on carrot seed was killed by soaking the seed in hot water (52°C for 25 min), and a subsequent PMA treatment of these hot-water-treated seed washes suppressed detection of the pathogen with both the real-time PCR and LAMP assays. For 36 commercial seed lots treated with PMA but not hot water, regression of colony counts of X. hortorum pv. carotae measured by dilution plating on a semiselective agar medium versus estimates of pathogen CFU determined by the molecular assays resulted in significant (P ≤ 0.05) linear relationships (R2 = 0.68 for the real-time PCR assay and 0.79 for the LAMP assay). The molecular assays provided quantitative estimates of X. hortorum pv. carotae infestations in carrot seed lots in <24 h, which is a significant improvement over the 7 to 14 days required to obtain results from the traditional dilution-plating assay.

scholarly journals Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Alexandre R. Vieira ◽  
Kathleen B. Deeley ◽  
Nicholas F. Callahan ◽  
Jacqueline B. Noel ◽  
Ida Anjomshoaa ◽  
...  
Keyword(s):  
Real Time ◽  
Streptococcus Mutans ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Bacterial Colonization ◽  
Human Saliva ◽  
Caries Experience ◽  
Multifactorial Disease ◽  
Pcr Assay ◽  
Human Dna

Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults.

scholarly journals A Real-Time, Quantitative PCR Seed Assay for Botrytis spp. that Cause Neck Rot of Onion

Plant Disease ◽  
2007 ◽  
Vol 91 (5) ◽  
pp. 599-608 ◽  
Author(s):  
Martin I. Chilvers ◽  
Lindsey J. du Toit ◽  
Hajime Akamatsu ◽  
Tobin L. Peever
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Intergenic Spacer ◽  
Fungal Species ◽  
Pcr Assay ◽  
The Real ◽  
Real Time Quantitative Pcr ◽  
Onion Seed ◽  
Pure Cultures ◽  
Fluorescent Polymerase Chain Reaction

A real-time fluorescent polymerase chain reaction (PCR) assay was developed using SYBR Green chemistry to quantify the Botrytis spp. associated with onion (Allium cepa) seed that are also able to induce neck rot of onion bulbs, i.e., B. aclada, B. allii, and B. byssoidea. The nuclear ribosomal intergenic spacer (IGS) regions of target and nontarget Botrytis spp. were sequenced, aligned, and used to design a primer pair specific to B. aclada, B. allii, and B. byssoidea. Primers and amplification parameters were optimized to avoid amplifying the related species B. cinerea, B. porri, and B. squamosa, as well as Sclerotinia sclerotiorum and isolates of 15 other fungal species commonly found associated with onion seed. The primers reliably detected 10 fg of genomic DNA per PCR reaction extracted from pure cultures of B. aclada and B. allii. Conventional assays of surface-disinfested and nondisinfested seed on an agar medium were used to determine the incidence of neck rot Botrytis spp. associated with each of 23 commercial onion seed lots, and the real-time PCR assay was used to determine the quantity of DNA of neck rot Botrytis spp. in each seed lot. A linear relationship could not be found between the incidence of seed infected with the neck rot Botrytis spp. using the conventional agar seed assays and the quantity of DNA of the neck rot Botrytis spp. detected by the real-time PCR assay. However, the real-time PCR assay appeared to be more sensitive than the conventional agar assay, allowing detection of neck rot Botrytis spp. in 5 of the 23 seed lots that tested negative using the conventional agar seed assay.

scholarly journals Development of a multiplex real-time PCR assay for BCG and validation in a clinical laboratory

2021 ◽  
Author(s):  
Shannon Catherine Duffy ◽  
Manigandan Venkatesan ◽  
Shubhada Chothe ◽  
Indira Poojary ◽  
Valsan Philip Verghese ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Laboratory ◽  
Limit Of Detection ◽  
Infected Tissue ◽  
Nucleotide Polymorphisms ◽  
Pcr Assay ◽  
Live Attenuated Vaccine ◽  
Pure Cultures ◽  
Improve Patient Management

Mycobacterium bovis bacille Calmette-Guérin (BCG) is a live attenuated vaccine which can result in local or disseminated infection, most commonly in immunocompromised individuals. Differentiation of BCG from other members of the Mycobacterium tuberculosis complex (MTBC) is required to diagnose BCG disease, which requires specific management. Current methods for BCG diagnosis are based on mycobacterial culture and conventional PCR; the former is time-consuming and the latter often unavailable. Further, there are reports that certain BCG strains may be associated with a higher rate of adverse events. This study describes the development of a two-step multiplex real-time PCR assay which uses single nucleotide polymorphisms to detect BCG and identify early or late BCG strains. The assay has a limit of detection of 1 pg BCG boiled lysate DNA and was shown to detect BCG in both pure cultures and experimentally infected tissue. Performance was assessed on 19 suspected BCG clinical isolates at Christian Medical College in Vellore, India taken from January 2018 to August 2020. Of these 19 isolates, 10 were identified as BCG (6 early and 4 late strains) and 9 were identified as other MTBC members. Taken together, the results demonstrate the ability of this assay to identify and characterize BCG disease from cultures and infected tissue. The capacity to identify BCG may improve patient management and the ability to discriminate between BCG strains may enable BCG vaccine pharmacovigilance.

scholarly journals Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status

2017 ◽  
Vol 55 (11) ◽  
pp. 3201-3209 ◽  
Author(s):  
Sumudu R. Perera ◽  
Nurul H. Khan ◽  
Irene Martin ◽  
Ali Taheri ◽  
Rajinder P. Parti ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Simultaneous Identification ◽  
Susceptibility Status

ABSTRACTA real-time PCR (RT-PCR) assay was designed for the simultaneous identification ofNeisseria gonorrhoeaeand its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) ingyrAofN. gonorrhoeae. The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254N. gonorrhoeaeisolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non-N. gonorrhoeaespecies isolates. The performance of the primers was validated using genomic DNA from 100 differentN. gonorrhoeaeisolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non-N. gonorrhoeaeisolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of theN. gonorrhoeaeisolates and 23 non-N. gonorrhoeaeisolates. These primers detectedN. gonorrhoeaeand its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification ofN. gonorrhoeaeand its ciprofloxacin susceptibility status.

scholarly journals Establishment of a quadruplex real-time PCR assay to distinguish the fungal pathogens Diaporthe longicolla, D. caulivora, D. eres, and D. novem on soybean

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257225
Author(s):  
Behnoush Hosseini ◽  
Ralf T. Voegele ◽  
Tobias I. Link
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Economic Damage ◽  
Pcr Assay ◽  
Plant Materials ◽  
Fungal Plant Pathogens ◽  
Pure Cultures ◽  
Soybean Leaves

Diaporthe species are fungal plant pathogens of many important crops. Seed decay is one of the most important diseases on soybean. It is caused by various species of the genus Diaporthe and responsible for significant economic damage. In central Europe the four species D. longicolla, D. caulivora, D. eres, and D. novem are considered the principal species of Diaporthe on soybean. Fast and accurate detection of these pathogens is of utmost importance. In this study four species-specific TaqMan primer-probe sets that can be combined into a quadruplex assay were designed based on TEF sequences. The specificity and efficiency of the primer-probe sets were tested using PCR products and genomic DNA from pure cultures of the four Diaporthe species and other soybean fungal pathogens. Our results indicate that the primer-probe sets DPCL, DPCC, DPCE, and DPCN allow discrimination of D. longicolla, D. caulivora, D. eres, and D. novem, respectively, and can be used to detect and quantify these four Diaporthe species in parallel using quadruplex real-time PCR. In addition, the quadruplex real-time PCR assay was evaluated on different plant materials including healthy and infected soybean seeds or seed lots, soybean stems, and soybean leaves. This assay is a rapid and effective method to detect and quantify Diaporthe species from samples relevant for disease control.

Detection and differentiation of Vibrio parahaemolyticus by multiplexed real-time PCR

2018 ◽  
Vol 64 (11) ◽  
pp. 809-815 ◽  
Author(s):  
Deshun Xu ◽  
Lei Ji ◽  
Xiaofang Wu ◽  
Wei Yan ◽  
Liping Chen
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Virulence Genes ◽  
Annealing Temperature ◽  
Cross Reactivity ◽  
Bacterial Strains ◽  
Pcr Assay ◽  
Pure Cultures ◽  
Sporadic Cases

Vibrio parahaemolyticus is a common and important pathogen that causes human gastroenteritis worldwide. A rapid, sensitive, and specific assay is urgently required for detection and differentiation of V. parahaemolyticus strains. We designed three sets of primers and probes using groEL and two virulence genes (tdh and trh) from V. parahaemolyticus, and developed a multiplex real-time PCR protocol. The sensitivity and specificity of the multiplex assay was evaluated by environmental and clinical specimens of V. parahaemolyticus. The multiplex PCR response system and annealing temperature were optimized. The detection limits of the multiplex real-time PCR were 104 and 105 CFU/mL (or CFU/g) in pure cultures and spiked oysters, respectively. The multiplex real-time PCR specifically detected and differentiated V. parahaemolyticus from 35 Vibrio strains and 11 other bacterial strains. Moreover, this method can detect and distinguish virulent from nonvirulent strains, with no cross-reactivity observed in the bacteria tested. This newly established multiplex real-time PCR assay offers rapid, specific, and reliable detection of the total and pathogenic V. parahaemolyticus strains, which is very useful during outbreaks and sporadic cases caused by V. parahaemolyticus infection.

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