Conventional and real-time PCR assays for specific detection and quantification of Fusarium oxysporum f. sp. ciceris in plants using intergenic spacer region-based marker

Biologia ◽  
2015 ◽  
Vol 70 (3) ◽  
Author(s):  
Kumari Priyanka ◽  
Sunil C. Dubey ◽  
Arun K. Singh
Keyword(s):  
Fusarium Oxysporum ◽  
Host Plant ◽  
Real Time ◽  
Real Time Pcr ◽  
Infected Plant ◽  
Intergenic Spacer ◽  
Infected Host ◽  
Pcr Assay ◽  
Intergenic Spacer Region ◽  
Plant Samples

AbstractFusarium wilt of chickpea, caused by Fusarium oxysporum f. sp. ciceris (Foc) is one of the most important fungal diseases worldwide. The detection of the pathogen at reasonable time period is of great importance, which requires rapid and sensitive detection methods. The intraspecific divergence sequences found in the intergenic spacer region (IGS) were selected and utilized with the aim to develop a molecular marker specifically to identify the Foc. A marker set, ISR52 F1 and R1 developed, was tested for their specificity as well as sensitivity using conventional as well as real-time polymerase chain reaction (PCR). The specificity of the marker was tested against Foc, other Fusarium species which are closely related to Foc as well as with artificially infected host plant samples. The detection limits of conventional PCR assay was up to 100 pg of infected plant DNA. It proved possible to amplify the IGS region in different portion of a Foc infected host plant by this PCR method. Furthermore, the real-time assay showed more sensitivity and was able to detect the pathogen in infected chickpea plant samples at the DNA concentration of 5 pg. A single melting peak obtained at 87.5°C showed the specificity of the marker towards Foc. Thus, real-time PCR assay proved their potentiality for same-day diagnosis of fungal infection and can be used as a rapid and effective procedure for routine detection and identification of Foc in chickpea samples.

scholarly journals Characterization of a novel Mycoplasma cynos real-time PCR assay

2019 ◽  
Vol 32 (6) ◽  
pp. 793-801 ◽  
Author(s):  
Rebecca L. Tallmadge ◽  
Renee Anderson ◽  
Patrick K. Mitchell ◽  
Zachary C. Forbes ◽  
Brenda Werner ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Intergenic Spacer ◽  
High Specificity ◽  
Mycoplasma Species ◽  
Limit Of Quantification ◽  
Clinical Specimens ◽  
Intergenic Spacer Region ◽  
Tuf Gene ◽  
Gene Assay

Mycoplasma cynos is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3–97.9% ( r2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1–122.5% ( r2 = 0.9784). The assay performed very well over a range of template input, from 109 copies to the lower limit of quantification at 4 copies of the M. cynos genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.

scholarly journals Development and Evaluation of a TaqMan Real-Time PCR Assay for Fusarium oxysporum f. sp. spinaciae

Plant Disease ◽  
2013 ◽  
Vol 97 (7) ◽  
pp. 927-937 ◽  
Author(s):  
P. A. Okubara ◽  
L. A. Harrison ◽  
E. W. Gatch ◽  
G. Vandemark ◽  
K. L. Schroeder ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Real Time ◽  
Seed Production ◽  
Fusarium Wilt ◽  
Real Time Pcr ◽  
Soil Samples ◽  
Soilborne Pathogen ◽  
Pcr Assay ◽  
The Real ◽  
Pathogen Dna

Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the United States suitable for commercial spinach seed production. A TaqMan real-time polymerase chain reaction (PCR) assay was developed for rapid identification and quantification of the pathogen, based on sequencing the intergenic spacer (IGS) region of rDNA of isolates of the pathogen. A guanine single-nucleotide polymorphism (G SNP) was detected in the IGS sequences of 36 geographically diverse isolates of F. oxysporum f. sp. spinaciae but not in the sequences of 64 isolates representing other formae speciales and 33 isolates representing other fungal species or genera. The SNP was used to develop a probe for a real-time PCR assay. The real-time PCR assay detected F. oxysporum f. sp. spinaciae at 3–14,056 CFU/g of soil in 82 soil samples collected over 3 years from naturally infested spinach seed production sites in western Washington, although a reliable detection limit of the assay was determined to be 11 CFU/g of soil. A significant (P < 0.05), positive correlation between enumeration of F. oxysporum on Komada's agar and quantification of the pathogen using the TaqMan assay was observed in a comparison of 82 soil samples. Correlations between pathogen DNA levels, Fusarium wilt severity ratings, and spinach biomass were significantly positive for one set of naturally infested soils but not between pathogen DNA levels, wilt incidence ratings, and spinach biomass for other soil samples, suggesting that soilborne pathogen population is not the sole determinant of spinach Fusarium wilt incidence or severity. The presence of the G SNP detected in one isolate of each of F. oxysporum ff. spp. lageneriae, lilii, melongenae, and raphani and reaction of the real-time PCR assay with 16 of 22 nonpathogenic isolates of F. oxysporum associated with spinach plants or soil in which spinach had been grown potentially limits the application of this assay. Nonetheless, because all isolates of F. oxysporum f. sp. spinaciae tested positive with the real-time PCR assay, the assay may provide a valuable means of screening for resistance to Fusarium wilt by quantifying development of the pathogen in spinach plants inoculated with the pathogen.

New PCR-based assay for the identification of Pectobacterium carotovorum causing potato soft rot

Plant Disease ◽  
2021 ◽  
Author(s):  
M. Belén Suárez ◽  
Marta Diego ◽  
F. J. Feria ◽  
M J Martín-Robles ◽  
Sergio Moreno ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Intergenic Spacer ◽  
Soft Rot ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Accurate Identification ◽  
Intergenic Spacer Region

Soft rot on potato tuber is a destructive disease caused by pathogenic bacterial species of the genera Pectobacterium and Dickeya. Accurate identification of the causal agent is necessary to ensure adequate disease management, since different species may have distinct levels of aggressiveness and host range. One of the most important potato pathogens is P. carotovorum, a highly heterogeneous species capable of infecting multiple hosts. The complexity of this species, until recently divided into several subspecies, has made it difficult to develop precise diagnostic tests. This study proposes a PCR assay based on the new pair of primers Pcar1F/R to facilitate the identification of potato isolates of P. carotovorum according to the most recent taxonomic description of this species. The new primers were designed on a variable segment of the 16S rRNA gene and the intergenic spacer region (ITS) of available DNA sequences from classical and recently established species in the genus Pectobacterium. The results of the PCR analysis of genomic DNA from 32 Pectobacterium and Dickeya strains confirmed that the Pcar1F/R primers have sufficient nucleotide differences to discriminate between P. carotovorum and other Pectobacterium species associated with damage to potato crops, with the exception of P. versatile, which improves the specificity of the currently available primers. The proposed assay was originally developed as a conventional PCR but was later adapted to the real-time PCR format for application in combination with the existing real-time PCR test for the potato-specific pathogen P. parmentieri. This should be useful for the routine diagnosis of potato soft rot.

scholarly journals Application of Real-Time PCR for Quantification of Microcystin Genotypes in a Population of the Toxic Cyanobacterium Microcystis sp

2003 ◽  
Vol 69 (11) ◽  
pp. 6723-6730 ◽  
Author(s):  
Rainer Kurmayer ◽  
Thomas Kutzenberger
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Total Population ◽  
Intergenic Spacer ◽  
Threshold Level ◽  
Logarithmic Scale ◽  
Cycle Number ◽  
Intergenic Spacer Region ◽  
Cell Numbers ◽  
The Mean

ABSTRACT The cyanobacterium Microcystis sp. frequently develops water blooms consisting of organisms with different genotypes that either produce or lack the hepatotoxin microcystin. In order to monitor the development of microcystin (mcy) genotypes during the seasonal cycle of the total population, mcy genotypes were quantified by means of real-time PCR in Lake Wannsee (Berlin, Germany) from June 1999 to October 2000. Standard curves were established by relating cell concentrations to the threshold cycle (the PCR cycle number at which the fluorescence passes a set threshold level) determined by the Taq nuclease assay (TNA) for two gene regions, the intergenic spacer region within the phycocyanin (PC) operon to quantify the total population and the mcyB gene, which is indicative of microcystin synthesis. In laboratory batch cultures, the cell numbers inferred from the standard curve by TNA correlated significantly with the microscopically determined cell numbers on a logarithmic scale. The TNA analysis of 10 strains revealed identical amplification efficiencies for both genes. In the field, the proportion of mcy genotypes made up the smaller part of the PC genotypes, ranging from 1 to 38%. The number of mcyB genotypes was one-to-one related to the number of PC genotypes, and parallel relationships between cell numbers estimated via the inverted microscope technique and TNA were found for both genes. It is concluded that the mean proportion of microcystin genotypes is stable from winter to summer and that Microcystis cell numbers could be used to infer the mean proportion of mcy genotypes in Lake Wannsee.

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 Prevalence and phylogenetic analysis of Mycoplasma synoviae strains isolated from Polish chicken layer flocks

2019 ◽  
Vol 63 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Olimpia Kursa ◽  
Grzegorz Tomczyk ◽  
Anna Sawicka
Keyword(s):  
Phylogenetic Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Genetic Material ◽  
Clinical Signs ◽  
Intergenic Spacer ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Mycoplasma Synoviae ◽  
Intergenic Spacer Region

AbstractIntroduction:Mycoplasma synoviae(MS) is a chicken pathogen of major economic importance.Material and Methods:Between 2010 and 2016, 906 commercial layer chicken flocks in Poland were examined for MS, and the phylogenetic relationship among the strains was established. Regionally dispersed samples were collected and tested with the use of real-time PCR to detect the 16S–23S intergenic spacer region. Positive samples were also tested with LAMP and conventional PCR to detect thevlhAgene.Results:MS genetic material was detected in 265 (29%) of the tested flocks by real-time PCR, in 227 by the LAMP method and in 202 (22%) by conventional PCR. The by-year percentage of positive samples began at 34% in 2010, rose to 44% in 2012, and declined to 29% in 2016. A phylogenetic analysis of PolishM. synoviaestrains using a partial sequence of thevlhAgene showed nine genotypes (A–I), the most frequently occurring being F and C. Pathogenic Polish MS field isolates (n = 27) collected from chickens with clinical signs of infection were grouped for their characteristic symptoms: respiratory for genotypes C, E, F, and I (n = 13), EAA and a drop in laying for genotypes F, E, and C (n = 12), and synovitis for genotype A (n = 2).Conclusion:These data showed the country’s isolate diversity. The high prevalence suggests the need to introduce appropriate control programmes. This is the first report of molecular epidemiological data onM. synoviaeinfection in layer chickens in Poland.

scholarly journals Intergenic Spacer–RFLP Analysis and Direct Quantification of Australian Fusarium oxysporum f. sp. vasinfectum Isolates from Soil and Infected Cotton Tissues

Plant Disease ◽  
2007 ◽  
Vol 91 (12) ◽  
pp. 1564-1573 ◽  
Author(s):  
A. G. Zambounis ◽  
E. Paplomatas ◽  
A. S. Tsaftaris
Keyword(s):  
Fusarium Oxysporum ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Vascular System ◽  
Sequence Data ◽  
Intergenic Spacer ◽  
Detection Sensitivity ◽  
Cotton Production ◽  
Fungal Dna

Fusarium wilt of cotton, caused by Fusarium oxysporum f. sp. vasinfectum, can have devastating effects on the vascular system of cotton plants and is a major threat to cotton production throughout the world. Accurate characterization and improved detection of these pathogenic isolates is needed for the implementation of a disease prevention program and the development of disease management strategies. Polymerase chain reaction (PCR) amplification of the ribosomal intergenic spacer (IGS) regions combined with digestion with three restriction enzymes (AluI, HaeIII, RsaI) resulted in three unique restriction profiles (IGS-restriction fragment length polymorphism [RFLP] haplotypes) for Australian F. oxysporum f. sp. vasinfectum isolates, which were capable of distinguishing them from other formae speciales of F. oxysporum. Furthermore, a portion of the IGS region flanking the 5′ end was sequenced and single nucleotide polymorphisms (SNPs) were revealed. Using these sequence data, two specific real-time PCR-based assays were developed for the absolute quantification of genomic DNA from isolates obtained from soil substrates and infected cotton tissues. Standard curves of real-time PCR-based assays showed a linear relation (R2 = 0.993 to 0.994) between log values of fungal genomic DNA and real-time PCR cycle thresholds. Using these assays, it was possible to detect fungal DNA as low as 5 pg/μl. The detection sensitivity for inoculum added to sterile soils was lower than 104 conidia/g soil. In plant samples, the quantified fungal DNA varied from 30 pg to 1 ng/100 ng of total plant genomic DNA.

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