scholarly journals PCR-Based Strategy To Detect and Identify Species of Phaeoacremonium Causing Grapevine Diseases

2007 ◽  
Vol 73 (9) ◽  
pp. 2911-2918 ◽  
Author(s):  
Angeles Aroca ◽  
Rosa Raposo
Keyword(s):  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Its Region ◽  
Restriction Enzymes ◽  
Effective Measure ◽  
Petri Disease ◽  
Specific Pcr ◽  
Plant Nursery ◽  
Species Specific ◽  
Single Amplicon

ABSTRACT Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial β-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.

scholarly journals Authentication of the Herbal Medicine Angelicae Dahuricae Radix Using an ITS Sequence-Based Multiplex SCAR Assay

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2134 ◽  
Author(s):  
Pureum Noh ◽  
Wook Kim ◽  
Sungyu Yang ◽  
Inkyu Park ◽  
Byeong Moon
Keyword(s):  
Herbal Medicines ◽  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Its Region ◽  
Sequencing Analysis ◽  
Accurate Identification ◽  
Republic Of China ◽  
Sequence Characterized Amplified Region ◽  
Angelicae Dahuricae Radix ◽  
Species Specific

The accurate identification of plant species is of great concern for the quality control of herbal medicines. The Korean Pharmacopoeia and the Pharmacopoeia of the People’s Republic of China define Angelicae Dahuricae Radix (Baek-Ji in Korean and Bai-zhi in Chinese) as the dried roots of Angelica dahurica or A. dahurica var. formosana belonging to the family Apiaceae. Discrimination among Angelica species on the basis of morphological characteristics is difficult due to their extremely polymorphic traits and controversial taxonomic history. Furthermore, dried roots processed for medicinal applications are indistinguishable using conventional methods. DNA barcoding is a useful and reliable method for the identification of species. In this study, we sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes in A. dahurica, A. dahurica var. formosana, and the related species A. anomala and A. japonica. Using these sequences, we designed species-specific primers, and developed and optimized a multiplex sequence-characterized amplified region (SCAR) assay that can simply and rapidly identify respective species, and verify the contamination of adulterant depending on the polymerase chain reaction (PCR) amplification without sequencing analysis in a single PCR reaction. This assay successfully identified commercial samples of Angelicae Dahuricae Radix collected from Korean and Chinese herbal markets, and distinguished them from adulterants. This multiplex SCAR assay shows a great potential in reducing the time and cost involved in the identification of genuine Angelicae Dahuricae Radix and adulterant contamination.

A simple method for discriminating Bursaphelenchus xylophilus and B. mucronatus by species-specific polymerase chain reaction primer pairs

Nematology ◽  
2004 ◽  
Vol 6 (2) ◽  
pp. 273-277 ◽  
Author(s):  
Koji Matsunaga ◽  
Katsumi Togashi
Keyword(s):  
Pcr Amplification ◽  
Nematode Species ◽  
Bursaphelenchus Xylophilus ◽  
Polymerase Chain Reaction Primer ◽  
Dna Fragments ◽  
Simple Method ◽  
Specific Pcr ◽  
Pcr Products ◽  
Specific Pcr Primer ◽  
Species Specific

Abstract Two species-specific PCR primer pairs were developed for identifying the two nematode species, Bursaphelenchus xylophilus and B. mucronatus. The primer pairs were developed from the sequence of ribosomal DNA (rDNA) repeats to produce DNA fragments of different lengths by PCR amplification. The DNA fragments for B. mucronatus and B. xylophilus were 210 bp and 557 bp, respectively. When mixed, neither primer pair inhibited the PCR amplification of the other. Five isolates of B. xylophilus and four isolates of B. mucronatus showed different band profiles of PCR products between the two species, but identical profiles among isolates of the same species.

scholarly journals Detection of Pythium ultimum Using Polymerase Chain Reaction with Species-Specific Primers

Plant Disease ◽  
1997 ◽  
Vol 81 (10) ◽  
pp. 1155-1160 ◽  
Author(s):  
K. Kageyama ◽  
A. Ohyama ◽  
M. Hyakumachi
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Transcribed Spacer ◽  
Pcr Amplification ◽  
Its Region ◽  
Pythium Ultimum ◽  
Pythium Species ◽  
Chain Reaction ◽  
Specific Primers ◽  
Polymerase Chain ◽  
Species Specific

This study was conducted to sequence the rDNA internal transcribed spacer (ITS) region of Pythium ultimum and Pythium group HS, design species-specific primers for polymerase chain reaction (PCR), and detect P. ultimum from diseased seedlings using PCR. The sequence of the ITS region of P. ultimum was identical with that of Pythium group HS. The results support the reports that the HS group is an asexual strain of P. ultimum. Using PCR, the primer pair K1+K3, designed on portions of the sequence of the ITS region, amplified isolates of P. ultimum and the HS group but not isolates of 20 other Pythium species. DNA extracts from damped-off seedlings were not amplified, but a 10-fold dilution of the extracts with Tris-EDTA (TE) buffer diluted the inhibitors and allowed PCR amplification. The primer pair used detected P. ultimum from a single diseased seedling.

A Variable 23S rDNA Region is a Useful Discriminating Target for Genus-Specific and Species-Specific PCR Amplification in Arcobacter Species

1995 ◽  
Vol 18 (3) ◽  
pp. 353-356 ◽  
Author(s):  
Katrin Bastyns ◽  
Dagmar Cartuyvels ◽  
Sabine Chapelle ◽  
Peter Vandamme ◽  
Herman Goossens ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Specific Pcr ◽  
Species Specific

Specific PCR Detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in Chicken Meat

2009 ◽  
Vol 72 (7) ◽  
pp. 1491-1495 ◽  
Author(s):  
DANIELA PENTIMALLI ◽  
NICOLETTE PEGELS ◽  
TERESA GARCÍA ◽  
ROSARIO MARTÍN ◽  
ISABEL GONZÁLEZ
Keyword(s):  
Pcr Amplification ◽  
Chicken Meat ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Gyra Gene ◽  
Specific Primers ◽  
Specific Pcr ◽  
Arcobacter Butzleri ◽  
Species Specific

An enrichment PCR assay using species-specific primers was developed for the detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in chicken meat. Primers for A. cryaerophilus, A. skirrowii, and A. cibarius were designed based on the gyrA gene to amplify nucleic acid fragments of 212, 257, and 145 bp, respectively. The A. butzleri–specific primers were designed flanking a 203-bp DNA fragment in the 16S rRNA gene. The specificity of the four primer pairs was assessed by PCR analysis of DNA from a panel of Arcobacter species, related Campylobacter, Helicobacter species, and other food bacteria. The applicability of the method was then validated by testing 42 fresh retail-purchased chicken samples in the PCR assay. An 18-h selective preenrichment step followed by PCR amplification with the four Arcobacter primer sets revealed the presence of Arcobacter spp. in 85.7% of the retail chicken samples analyzed. A. butzleri was the only species present in 50% of the samples, and 35.7% of the samples were positive for both A. butzleri and A. cryaerophilus. A. skirrowii and A. cibarius were not detected in any of the chicken samples analyzed. The enrichment PCR assay developed is a specific and rapid alternative for the survey of Arcobacter contamination in meat.

scholarly journals Species-Specific PCR Assays for Differentiating Heterodera filipjevi and H. avenae

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1611-1619 ◽  
Author(s):  
Guiping Yan ◽  
Richard W. Smiley ◽  
Patricia A. Okubara ◽  
Andrea M. Skantar
Keyword(s):  
Pacific Northwest ◽  
Management Practices ◽  
Pcr Amplification ◽  
Nematode Species ◽  
Heterodera Avenae ◽  
Cyst Nematodes ◽  
Specific Pcr ◽  
The Pacific ◽  
Pcr Assays ◽  
Species Specific

Heterodera avenae and H. filipjevi are economically important cyst nematodes that restrict production of cereal crops in the Pacific Northwest United States and elsewhere in the world. Identification of these two species is critical for recommending and implementing effective management practices. Primers were designed from the internal transcribed spacer (ITS) regions of H. avenae and H. filipjevi ribosomal DNA. The primers were highly specific when examined on target isolates but did not amplify DNA from nontarget Heterodera, Globodera, Meloidogyne, Pratylenchus, and other nematode species tested. Polymerase chain reaction (PCR) and amplification conditions were established, and H. avenae and H. filipjevi were clearly distinguished by PCR fragments of 242 and 170 bp, respectively. Robust PCR amplification was achieved with DNA extracted from a single egg or second-stage juvenile (J2) using a laboratory-made worm lysis buffer, and DNA from 0.5 egg or J2 using a commercial kit. The PCR assays were successfully employed for differentiation of H. filipjevi and H. avenae populations collected from eight locations in three Pacific Northwest states. This is the first report of a species-specific ITS PCR assay to detect and identify H. filipjevi. The assays for both species will enhance diagnosis of cereal cyst nematode species in infested fields.

scholarly journals Species-Specific Plasmid Sequences for PCR Identification of the Three Species of Borrelia burgdorferiSensu Lato Involved in Lyme Disease

1998 ◽  
Vol 36 (1) ◽  
pp. 269-272 ◽  
Author(s):  
Marie-Christine Misonne ◽  
Philippe Pierre Hoet
Keyword(s):  
Lyme Disease ◽  
Pcr Amplification ◽  
Sensu Stricto ◽  
Pcr Assay ◽  
Borrelia Garinii ◽  
Specific Pcr ◽  
Pcr Identification ◽  
Identification Of Species ◽  
Species Specific ◽  
Specific Sequences

Species-specific sequences were shown to be carried by plasmids of the three main species of Borrelia burgdorferi sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids of B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, respectively, were then built and used to isolate species-specific sequences. After sequencing of the cloned inserts, three sets of primers were designed. They were shown to determine species-specific PCR amplification products. The sensitivities of the PCR assay with these primers were 100 spirochetes for B. burgdorferi sensu stricto and 1,000 spirochetes for B. garinii and B. afzelii. The usefulness of these primers for the identification of species in biological samples (tick, serum, and cerebrospinal fluid samples) was ascertained.

scholarly journals Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

2015 ◽  
Vol 53 (10) ◽  
pp. 3280-3285 ◽  
Author(s):  
Sarah A. Ahmed ◽  
Wendy W. J. van de Sande ◽  
Marie Desnos-Ollivier ◽  
Ahmed H. Fahal ◽  
Najwa A. Mhmoud ◽  
...  
Keyword(s):  
Morphological Characteristics ◽  
High Specificity ◽  
Its Region ◽  
Molecular Techniques ◽  
Isothermal Amplification ◽  
Content Type ◽  
Fungal Dna ◽  
Madurella Mycetomatis ◽  
Phenotypic Methods ◽  
Species Specific

Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide isMadurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region ofM. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection ofM. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples.

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