scholarly journals Development of SCAR Primers for PCR Assay to Detect Diplodia seriata

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
M. T. Martín ◽  
M. J. Cuesta ◽  
L. Martín
Keyword(s):  
Random Amplified Polymorphic Dna ◽  
Fungal Species ◽  
Dna Purification ◽  
Pcr Assay ◽  
Diplodia Seriata ◽  
Castilla Y Leon ◽  
Target Dna ◽  
Pcr Products ◽  
Scar Primer ◽  
Rapd Fragment

The aim of this study was to develop primer pairs for Diplodia seriata identification, one of the most common fungal species associated with grapevine decline in Castilla y León (Spain). Genetic variability of selected isolates of D. seriata was estimated. A molecular marker was generated from a random amplified polymorphic DNA (RAPD) fragment. PCR products of around 1200 bp were obtained with OPE20 primer. The PCR products were cloned and sequenced. The sequences were compared and a fragment of 1207 bp was used to design primer pairs. Two primer pairs were selected (DS3.8 S3-DS3.8 R6 and DS3.8 S3-DS3.8 R4) that amplified a single DNA product of 634 bp and 233 bp, respectively, with D. seriata isolates. No amplification was obtained for any of the 57 isolates of other species. The designed SCAR primer pairs allowed a rapid detection of D. seriata, and were able to detect 0.1 pg of the target DNA. Detection was specific and sensitive for D. seriata. The established protocols detected these fungi in naturally infected grapevines after DNA purification. Diplodia seriata was detectable without DNA purification and isolation in 62.5% to 75% of reactions. The detection of this pathogen in wood samples has great potential for use in pathogen-free certification schemes.

A PCR Assay Based on a Sequence-Characterized Amplified Region Marker for Detection of Emetic Bacillus cereus

2004 ◽  
Vol 67 (8) ◽  
pp. 1694-1701 ◽  
Author(s):  
SHIGERU NAKANO ◽  
HIDEKI MAESHIMA ◽  
ATSUSHI MATSUMURA ◽  
KATSUTOSHI OHNO ◽  
SHIGEKO UEDA ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Enrichment Culture ◽  
Random Amplified Polymorphic Dna ◽  
Agar Plate ◽  
Brain Heart Infusion ◽  
Pcr Assay ◽  
Bacterial Colony ◽  
Sequence Characterized Amplified Region ◽  
Rapd Fragment ◽  
Bacillus Strains

A PCR assay for the detection of Bacillus cereus strains able to produce an emetic toxin (cereulide) was developed in this study based on a sequence-characterized amplified region (SCAR) derived from a random amplified polymorphic DNA (RAPD) fragment. One of the RAPD fragments generated was selected, cloned, and sequenced. A set of PCR primers was newly designed from the SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay. To determine the specificity of the assay, 30 different B. cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested. Results were positive for every emetic B. cereus strain and for only one nonemetic B. cereus strain. For all other bacterial strains, results were negative. Bacterial DNA for PCR was prepared by a simple procedure using Chelex 100 resin from the bacterial colony on the agar plate or from culture after growth in brain heart infusion medium. This PCR assay enabled us to detect the bacteria of emetic B. cereus grown on agar plates but not the bacteria of nonemetic B. cereus. To test this PCR assay for the monitoring of the emetic bacteria, 10 to 70 CFU of B. cereus DSM 4312 (emetic) per g of food was inoculated into several foods as an indicator, followed by a 7-h enrichment culture step. Because this PCR assay based on the SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of emetic B. cereus.

scholarly journals Development of a SCAR Marker-Based Diagnostic Method for the Detection of the Citrus Target Spot Pathogen Pseudofabraea citricarpa

2018 ◽  
Vol 2018 ◽  
pp. 1-5 ◽  
Author(s):  
Yuheng Yang ◽  
Junhua Hu ◽  
Fajing Chen ◽  
Dekuan Ding ◽  
Changyong Zhou
Keyword(s):  
Scar Marker ◽  
Random Amplified Polymorphic Dna ◽  
Fungal Species ◽  
Economic Losses ◽  
Sequence Characterized Amplified Region ◽  
Target Spot ◽  
Target Dna ◽  
Highly Sensitive ◽  
Citrus Production ◽  
Citrus Disease

Target spot, a recently observed citrus disease that is caused by Pseudofabraea citricarpa, can cause substantial economic losses in citrus production. In this study, a 797 bp marker specific to Ps. citricarpa was identified via random amplified polymorphic DNA (RAPD) technique. The primer pair Pc-SFP/Pc-SRP, which was designed from RAPD amplicons, was utilized as a sequence-characterized amplified region (SCAR) marker. This marker identified Ps. citricarpa with a single and distinct band of 389 bp but did not amplify DNA from other tested fungal species. The PCR assay was highly sensitive to the target DNA at picogram levels and could reliably amplify Ps. citricarpa sequences with the Pc-SFP/Pc-SRP primer pair. The SCAR marker that was identified in the present study can facilitate rapid decision-making and precise disease forecasting and management.

Variation in bacterial endosymbionts associated with the date palm hopper,Ommatissus lybicuspopulations

2017 ◽  
Vol 108 (2) ◽  
pp. 271-281 ◽  
Author(s):  
S. Karimi ◽  
H. Izadi ◽  
M. Askari Seyahooei ◽  
A. Bagheri ◽  
P. Khodaygan
Keyword(s):  
Date Palm ◽  
Multiple Infections ◽  
Infection Frequency ◽  
Pcr Assay ◽  
Specific Primers ◽  
Consensus Sequences ◽  
Pcr Products ◽  
Bacterial Endosymbionts ◽  
Different Populations ◽  
Polymerase Chain

AbstractThe date palm hopper,Ommatissus lybicus, is a key pest of the date palm, which is expected to be comprised of many allopatric populations. The current study was carried out to determine bacterial endosymbiont diversity in the different populations of this pest. Ten date palm hopper populations were collected from the main date palm growing regions in Iran and an additional four samples from Pakistan, Oman, Egypt and Tunisia for detection of primary and secondary endosymbionts using polymerase chain reaction (PCR) assay with their specific primers. The PCR products were directly sequenced and edited using SeqMan software. The consensus sequences were subjected to a BLAST similarity search. The results revealed the presence of ‘CandidatusSulcia muelleri’ (primary endosymbiont) andWolbachia,ArsenophonusandEnterobacter(secondary endosymbionts) in all populations. This assay failed to detect ‘CandidatusNasuia deltocephalinicola’ andSerratiain these populations. ‘Ca. S. muelleri’ exhibited a 100% infection frequency in populations andWolbachia,ArsenophonusandEnterobacterdemonstrated 100, 93.04 and 97.39% infection frequencies, respectively. The infection rate ofArsenophonusandEnterobacterranged from 75 to 100% and 62.5 to 100%, respectively, in different populations of the insect. The results demonstrated multiple infections by ‘Ca. Sulcia muelleri’,Wolbachia,ArsenophonusandEnterobacterin the populations and may suggest significant roles for these endosymbionts on date palm hopper population fitness. This study provides an insight to endosymbiont variation in the date palm hopper populations; however, further investigation is needed to examine how these endosymbionts may affect host fitness.

scholarly journals Interlaboratory Study on Thermal Cycler Performance in Controlled PCR and Random Amplified Polymorphic DNA Analyses

2001 ◽  
Vol 47 (1) ◽  
pp. 47-55 ◽  
Author(s):  
Ginny C Saunders ◽  
Juliet Dukes ◽  
Helen C Parkes ◽  
Johanne H Cornett
Keyword(s):  
Random Amplified Polymorphic Dna ◽  
Positive Sample ◽  
Interlaboratory Study ◽  
Pcr Assay ◽  
Single Product ◽  
The United Kingdom ◽  
Rapd Pcr ◽  
Block Temperature ◽  
Thermal Cycler ◽  
Correct Target

Abstract Background: Intercomparisons of PCR-based data between laboratories require an assurance of assay reproducibility. We performed an interlaboratory study to investigate the contribution made by a variety of thermal cyclers to PCR performance as measured by interblock reproducibility and intrablock repeatability. Methods: Two standardized assays designed to minimize the introduction of non-thermal-cycler-dependent variations were evaluated by 18 laboratories in the United Kingdom, using 33 thermal cyclers of various makes and models. We used a single-product (590 bp) PCR, established in our laboratory as a robust and specific reaction. The second reaction, a multiproduct random amplified polymorphic DNA (RAPD) PCR, was known to be more susceptible to small changes in block temperature and was therefore considered a way of assessing block uniformity with respect to temperature. Assay repeatability data were analyzed with respect to temperature calibration status, the type of temperature control mechanism, thermal cycler age, and the presence of oil overlay or heated lid systems. Results: All (100%) of the laboratories produced the correct target for the single-product PCR assay, although substantial variation in yield in replicate reactions was observed in 9.4% of these. The RAPD reaction generated results that varied extensively both within the same block and between different thermal cyclers. For eight replicates of a positive sample, 88% intrablock repeatability was demonstrated in calibrated thermal cyclers, which decreased to 63% in noncalibrated instruments. Conclusions: Irrespective of the make and model of thermal cycler, temperature-calibrated instruments consistently generated more repeatable RAPD data than noncalibrated instruments. Guidelines are offered on optimizing and monitoring thermal cycler performance.

scholarly journals Detection and Discrimination of Pratylenchus neglectus and P. thornei in DNA Extracts from Soil

Plant Disease ◽  
2008 ◽  
Vol 92 (11) ◽  
pp. 1480-1487 ◽  
Author(s):  
Guiping Yan ◽  
Richard W. Smiley ◽  
Patricia A. Okubara ◽  
Andrea Skantar ◽  
Sandra A. Easley ◽  
...  
Keyword(s):  
Nematode Species ◽  
Wheat Root ◽  
Fungal Species ◽  
Parasitic Nematodes ◽  
Rrna Gene ◽  
28S Rrna ◽  
Pratylenchus Neglectus ◽  
Pcr Products ◽  
Species Specific ◽  
Plant Parasitic

A species-specific polymerase chain reaction (PCR) method was developed to detect and identify the root-lesion nematodes Pratylenchus neglectus and P. thornei from soil. A primer set was designed from Pratylenchus 28S rRNA gene sequences of the D3 expansion domain. Primer specificity was confirmed with 23 isolates of 15 nematode species and other plant-parasitic and non-plant-parasitic nematodes typically present in the soil communities, and with six fungal species commonly associated with wheat root rot. DNA obtained using a commercially available kit and a method developed in our laboratory gave comparable amplification. PCR conditions were optimized and the two species were differentiated by PCR products of 144 bp for P. neglectus and 288 bp for P. thornei. With this assay, we detected a single juvenile in 1 g of sterile, inoculated soil. Examination of 30 field soil samples revealed that this method was applicable to a range of soils naturally infested with these two pathogens in Oregon. This PCR-based method is rapid, efficient, and reliable, does not require expertise in nematode taxonomy and morphology, and could be used as a rapid diagnostic tool for commercial and research applications for disease forecasting and management.

scholarly journals Isolation and Genotyping of Acanthamoeba from Soil Samples in Markazi Province, Iran

2018 ◽  
Vol 6 (12) ◽  
pp. 2290-2294 ◽  
Author(s):  
Mehri Meighani ◽  
Zahra Eslamirad ◽  
Reza Hajihossein ◽  
Azam Ahmadi ◽  
Sassan Saki
Keyword(s):  
Water Sources ◽  
Soil Samples ◽  
Pcr Assay ◽  
Predominant Genotype ◽  
Molecular Assays ◽  
Specific Medium ◽  
Pcr Products

AIM: A previous study confirmed the contamination of water sources with this parasite in Arak, Markazi Province, Iran. The current study investigated soil sources and determined the predominant genotype of Acanthamoeba in this region of Iran. MATERIAL AND METHODS: Forty-eight soil samples, collected from different regions of Arak, Markazi province, Iran, were evaluated in this study. The samples were processed and identified by culturing on a specific medium, performing PCR assay, and sequencing the PCR products. Finally, using the NCBI database, the genotypes were determined. RESULTS: Of 48 soil samples, 33.3% and 31.25% were contaminated with Acanthamoeba according to the culture and molecular assays, respectively. The majority of these isolates belonged to the T4, T5 and T6 genotypes of Acanthamoeba. CONCLUSION: The genotypes of most isolates from soil samples in Arak similar to other regions of Iran belong to T4 genotype of this parasite. New sequence accession numbers include MG066681 and MG298785-MG298794.  

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 Genetic diversity of fruit borer, Helicoverpa armigera (Lepidoptera: Noctuidae) based on random amplified polymorphic DNA- polymerase chain reaction

2014 ◽  
Vol 39 (2) ◽  
pp. 263-271
Author(s):  
AKMZ Rahman ◽  
MA Haque ◽  
SN Alam ◽  
P Yasodha ◽  
V Balasubramani
Keyword(s):  
Genetic Variability ◽  
Helicoverpa Armigera ◽  
Indian Population ◽  
Random Amplified Polymorphic Dna ◽  
Similarity Coefficient ◽  
Taq Polymerase ◽  
Agroecological Zones ◽  
Pcr Products ◽  
Helicoverpa Armígera ◽  
Fruit Borer

The genetic variability of Helicoverpa armigera (Hübner) at different agroecological zones of Bangladesh in comparison with Indian population was conducted in India during September 2008 to February 2009. A total of 12 H. armigera populations of which 10 populations collected from different agroecological zones of Bangladesh and two populations from India were tested for their genetic variability. Eight out of the ten primers produced scorable PCR products by amplifying the template DNA with taq polymerase and were subjected for analysis. Those eight primers got amplified to a total of 138 markers which produced polymorphic markers. The similarity coefficient based on 138 RAPD markers ranged from 0.000 to 0.777 of the pair-wise combination among twelve samples of H. armigera. An UPGMA dendrogram based on Jaccard’s similarity coefficient was constructed for the 12 samples of H. armigera. The dendrogram showed that H. armigera population from Bangladesh had 25 to 45 percent similarity, and in its Indian population the similarity remained within this range. DOI: http://dx.doi.org/10.3329/bjar.v39i2.20428 Bangladesh J. Agril. Res. 39(2): 263-271, June 2014

scholarly journals A PCR-based Technique for Identification of Fusicoccum sp. from Pistachio and Various Other Hosts in California

Plant Disease ◽  
2002 ◽  
Vol 86 (5) ◽  
pp. 515-520 ◽  
Author(s):  
Zhonghua Ma ◽  
Themis J. Michailides
Keyword(s):  
Host Plants ◽  
Extraction Procedure ◽  
Its Region ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Fungal Species ◽  
Pcr Assay ◽  
Shoot Blight ◽  
Dna Fragment ◽  
Polymerase Chain

Botryosphaeria panicle and shoot blight of pistachio, caused by Fusicoccum sp. is a destructive disease in California. In this study, a pair of group-specific polymerase chain reaction (PCR) primers BDI and BDII, was developed for identification of Fusicoccum sp. from pistachio and other hosts in California based on the sequences of the rDNA internal transcribed spacer (ITS) region. The primers amplified a 356-bp DNA fragment for all 73 tested isolates of Fusicoccum sp. collected from pistachio and other hosts throughout California in different years, but not for the other 33 fungal species isolated from pistachio and the eight isolates of Fusicoccum sp. obtained from pistachio trees in Greece. The PCR assay using this pair of primers was sensitive enough to detect 5 pg of genomic DNA of Fusicoccum sp. A simple DNA extraction procedure was developed that led to the rapid identification of Fusicoccum sp. from pistachio and other host plants in California.

scholarly journals Using the TxtAB Operon to Quantify Pathogenic Streptomyces in Potato Tubers and Soil

2008 ◽  
Vol 98 (4) ◽  
pp. 405-412 ◽  
Author(s):  
Xinshun Qu ◽  
Leslie A. Wanner ◽  
Barbara J. Christ
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Common Scab ◽  
Cost Effective ◽  
Potato Tubers ◽  
Pcr Assay ◽  
Streptomyces Species ◽  
Standard Curve ◽  
Target Dna

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R2 = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 101 to 106 pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 103 to 106 per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.

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