A Universal Microarray Detection Method for Identification of Multiple Phytophthora spp. Using Padlock Probes

The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5′ and 3′ ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.

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Detection and Identification of Fish-PathogenicAphanomyces piscicidaUsing Polymerase Chain Reaction (PCR) with Species-Specific Primers

Journal of Aquatic Animal Health ◽

10.1577/h03-047.1 ◽

2004 ◽

Vol 16 (4) ◽

pp. 220-230 ◽

Cited By ~ 20

Author(s):

Panarat Phadee ◽

Osamu Kurata ◽

Kishio Hatai ◽

Ikuo Hirono ◽

Takashi Aoki

Keyword(s):

Polymerase Chain Reaction ◽

Chain Reaction ◽

Specific Primers ◽

Detection And Identification ◽

Polymerase Chain ◽

Species Specific

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A Multiplex PCR for the Detection of Phytophthora nicotianae and P. cactorum, and a Survey of Their Occurrence in Strawberry Production Areas of Japan

Plant Disease ◽

10.1094/pdis-01-11-0076 ◽

2011 ◽

Vol 95 (10) ◽

pp. 1270-1278 ◽

Cited By ~ 28

Author(s):

Mingzhu Li ◽

Takahiro Asano ◽

Haruhisa Suga ◽

Koji Kageyama

Keyword(s):

Multiplex Pcr ◽

Protein Gene ◽

Phytophthora Nicotianae ◽

Soilborne Pathogens ◽

Chain Reaction ◽

Specific Primers ◽

Phytophthora Spp ◽

Polymerase Chain ◽

Species Specific ◽

Production Areas

We aimed to simultaneously detect two pathogens causing strawberry diseases, Phytophthora nicotianae and P. cactorum, by multiplex polymerase chain reaction (PCR), and to survey their occurrence in the main strawberry production areas of Japan. Due to the need to combine different primer pairs for multiplex PCR and the low specificity of published specific primers for P. nicotianae and P. cactorum, new species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions of ribosomal DNA and the ras-related protein gene Ypt1, respectively. Specificity of the designed primers was demonstrated using 68 isolates, including Phytophthora spp., Pythium spp., and other soilborne pathogens. Multiplex PCR discriminated between P. nicotianae and P. cactorum in DNA mixtures of mycelia of the two species. Moreover, both species were detected in artificially and naturally infested soils, indicating that these markers can be used in diagnosis of strawberry diseases. For investigation of the geographic distribution of the two pathogens in Japan, soil samples were collected in 89 strawberry fields from eight prefectures (Gifu, Saga, Nara, Tochigi, Chiba, Shizuoka, Yamanashi, and Hokkaido) of Japan. The method that was developed was successfully applied to survey P. nicotianae and P. cactorum, and distribution of the two pathogens in strawberry plantings in Japan was determined.

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Detection and Identification of Four Common Rust Pathogens of Cereals and Grasses Using Real-Time Polymerase Chain Reaction

Phytopathology ◽

10.1094/phyto-97-6-0717 ◽

2007 ◽

Vol 97 (6) ◽

pp. 717-727 ◽

Cited By ~ 55

Author(s):

C. W. Barnes ◽

L. J. Szabo

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Internal Control ◽

Chain Reaction ◽

Detection And Identification ◽

Pasture Grasses ◽

Significant Yield ◽

Polymerase Chain ◽

Species Specific ◽

Rust Pathogens

Puccinia spp. are widespread pathogens of cereals and grasses that annually cause significant yield losses worldwide, especially in barley, oat, and wheat. Urediniospore morphology and early symptom development have limited usefulness for distinguishing Puccinia spp. Therefore, we developed real-time polymerase chain reaction assays for rapid detection of the four rust pathogen species, Puccinia graminis (Pers.:Pers.), P. striiformis (Westend.), P. triticina (Eriks.), and P. recondita (Roberge ex Desmaz.). Duplex assays were constructed for the nuclear rDNA gene, using the variable internal transcribed spacer 1 (ITS1) region to distinguish between species, and the conserved 28S region as an internal control. Species-specific ITS1 primer/probe sets were highly specific and could detect <1 pg of DNA. The species-specific primer/probe sets showed positive results over a linear range of DNA five orders of magnitude or greater. Specificity of the assays was tested using multiple collections representing a range of races and formae speciales within a species. Additionally, assay specificity was evaluated by testing a range of other grass rust pathogens, as well as other fungi. The 28S primer/probe combination was successful in detecting all Puccinia spp. tested within the duplex assays, validating the integrity of each assay. Finally, the assays were used to identify unknown rust fungi infecting pasture grasses.

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Rapid Detection and Identification of Lactobacillus spp. in Dairy Products by Using the Polymerase Chain Reaction

Journal of Food Protection ◽

10.4315/0362-028x-59.10.1031 ◽

1996 ◽

Vol 59 (10) ◽

pp. 1031-1036 ◽

Cited By ~ 64

Author(s):

MARYANNE DRAKE ◽

CHRISTOPHER L. SMALL ◽

KEMET D. SPENCE ◽

BARRY G. SWANSON

Keyword(s):

Polymerase Chain Reaction ◽

Dairy Products ◽

Lactobacillus Helveticus ◽

Lactobacillus Delbrueckii ◽

Lactobacillus Species ◽

Chain Reaction ◽

Bacterial Dna ◽

Detection And Identification ◽

Polymerase Chain ◽

Species Specific

Species-specific primers for use in the polymerase chain reaction (PCR) were designed to differentially amplify DNA from the common dairy lactobacillus species Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus helveticus, and Lactobacillus acidophilus. A method for rapid extraction of bacterial DNA from dairy products was developed. The sensitivity of bacterial DNA extraction from food and subsequent amplification by PCR was 100 cells total. Lactobacillus DNA was extracted and identified from commercial yoghurts, acidophilus milk, and cheeses. The methodology allows the presumptive identification of dairy lactobacilli in less than 6 hours.

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Routine identification of four sympatric species of calanoid copepods Pseudocalanus spp. in the Atlantic Arctic using a species-specific polymerase chain reaction

Journal of Oceanological Research ◽

10.29006/1564-2291.jor-2020.48(1).4 ◽

2020 ◽

Vol 48 (1) ◽

pp. 62-72

Author(s):

E. A. Ershova

Keyword(s):

Polymerase Chain Reaction ◽

Life Cycles ◽

The Arctic ◽

Relative Distribution ◽

Chain Reaction ◽

Specific Polymerase Chain Reaction ◽

Routine Identification ◽

Polymerase Chain ◽

Species Specific ◽

Plankton Communities

Сalanoid copepods of the genus Pseudocalanus play an important role in the plankton communities of the Arctic and boreal seas, often dominating in numbers and constituting a significant proportion of the biomass of zooplankton. Despite their high presence and significance in the shelf plankton communities, species-specific studies of the biology of these are significantly hampered by extremely small morphological differences between them, especially at the juvenile stages, at which they are virtually indistinguishable. In this paper, we describe a new, routine and low-cost molecular method for identifying all Pseudocalanus species found in the Atlantic sector of the Arctic: the Arctic P. acuspes, P. minutus and the boreal P. moultoni and P. elongatus, and apply it to describe the relative distribution of these species in four locations of the Arctic and sub-Arctic. With this method, species-specific polymerase chain reaction (ssPCR), mass identification of individuals of any developmental stage, including nauplii, is possible. This method can serve as an excellent tool for studying the species-specific biology of this group, describing their life cycles, as well as monitoring changes in Arctic marine ecosystems under the influence of changing climate.

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Conversion of sequence-characterized amplified region (SCAR) bands into high-throughput DNA markers based on RAPD technique for detection of the stem nematode Ditylenchus dipsaci in crucial plant hosts

Plant Soil and Environment ◽

10.17221/2226-pse ◽

2008 ◽

Vol 53 (No. 3) ◽

pp. 97-104 ◽

Cited By ~ 12

Author(s):

M. Zouhar ◽

M. Marek ◽

O. Douda ◽

J. Mazáková ◽

P. Ryšánek

Keyword(s):

Cost Effective ◽

Healthy Plant ◽

Host Preferences ◽

Sequence Characterized Amplified Region ◽

Detection And Identification ◽

Plant Hosts ◽

Cost Effective Technique ◽

Ditylenchus Dipsaci ◽

Species Specific ◽

Template Dna

Ditylenchus dipsaci, the stem nematode, is a migratory endoparasite of over 500 species of angiosperms. The main method of D. dipsaci control is crop rotation, but the presence of morphologically indistinguishable host races with different host preferences makes rotation generally ineffective. Therefore, a sensitive, rapid, reliable, as well as cost effective technique is needed for identification of D. dipsaci in biological samples. This study describes the development of species-specific pairs of PCR oligonucleotides for detection and identification of the D. dipsaci stem nematode in various plant hosts. Designed DIT-2 primer pair specifically amplified a fragment of 325 bp, while DIT-5 primer pair always produced a fragment of 245 bp in all D. dipsaci isolates. Two developed SCAR primer pairs were further tested using template DNA extracted from a collection of twelve healthy plant hosts; no amplification was however observed. The developed PCR protocol has proved to be quite sensitive and able to specifically detect D. dipsaci in artificially infested plant tissues.

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A Comparative Study of 5S rDNA Non-Transcribed Spacers in Elaeagnaceae Species

Plants ◽

10.3390/plants10010004 ◽

2020 ◽

Vol 10 (1) ◽

pp. 4

Author(s):

Oleg S. Alexandrov ◽

Olga V. Razumova ◽

Gennady I. Karlov

Keyword(s):

Polymerase Chain Reaction ◽

Molecular Markers ◽

Dna Markers ◽

5S Rdna ◽

Chain Reaction ◽

Closely Related Species ◽

Pcr Products ◽

Polymerase Chain ◽

Species Specific ◽

Shepherdia Canadensis

5S rDNA is organized as a cluster of tandemly repeated monomers that consist of the conservative 120 bp coding part and non-transcribed spacers (NTSs) with different lengths and sequences among different species. The polymorphism in the 5S rDNA NTSs of closely related species is interesting for phylogenetic and evolutional investigations, as well as for the development of molecular markers. In this study, the 5S rDNA NTSs were amplified with universal 5S1/5S2 primers in some species of the Elaeagnaceae Adans. family. The polymerase chain reaction (PCR) products of five Elaeagnus species had similar lengths near 310 bp and were different from Shepherdia canadensis (L.) Nutt. and Sh. argentea (Pusch.) Nutt. samples (260 bp and 215 bp, respectively). The PCR products were cloned and sequenced. An analysis of the sequences revealed that intraspecific levels of NTS identity are high (approximately 95–96%) and similar in the Elaeagnus L. species. In Sh. argentea, this level was slightly lower due to the differences in the poly-T region. Moreover, the intergeneric and intervarietal NTS identity levels were studied and compared. Significant differences between species (except E. multiflora Thunb. and E. umbellata Thunb.) and genera were found. Herein, a range of the NTS features is discussed. This study is another step in the investigation of the molecular evolution of Elaeagnaceae and may be useful for the development of species-specific DNA markers in this family.

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