5585238 Detection of fungal pathogens using the polymerase chain reaction

Diagnosis of yellow vine disease (YVD) in cucurbits, an important disease in the south-central United States, relies on external symptom appearance, phloem discoloration, and the presence of bacterium-like organisms (BLOs) in phloem. Polymerase chain reaction (PCR) amplification of BLO nucleotide sequences was explored as a means to improve diagnostic techniques. PCR, using a primer pair based on sequences of the citrus-greening BLO, amplified a 0.15-kilobase (kb) fragment from the DNA of symptomatic plants, but not from that of asymptomatic plants. Its nucleotide sequence suggested that the DNA amplified was of pro-karyotic origin. A primer pair, designed to amplify nonspecific prokaryotic 16S rDNA, amplified a 1.5-kb DNA fragment in both the symptomatic and asymptomatic plants. The 1.5-kb fragment from the asymptomatic plants corresponded to chloroplast 16S rDNA, and the band from the symptomatic plants was composed of 16S rDNAs from both chloroplasts and a prokaryote. The nucleotide sequence of the prokaryotic DNA was determined and used to design three primers (YV1, YV2, and YV3). Fragments of 0.64 and 1.43 kb were amplified with primers YV1-YV2 and primers YV1-YV3, respectively, from symptomatic plants. Neither primer set yielded fragments from asymptomatic plants, unrelated bacteria, or selected soilborne fungal pathogens of cucurbits. Phylogenetic analysis indicated that the prokaryote is a gamma-3 proteobacterium. The consistent association of the 0.64- and 1.43-kb fragments with symptomatic plants suggests that the gamma-3 proteobacterium may be the causal agent of YVD of cantaloupe, squash, and watermelon.

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Dematiaceous fungal pathogens: analysis of ribosomal DNA gene polymorphism by polymerase chain reaction-restriction fragment length polymorphism

Mycoses ◽

10.1046/j.1439-0507.1999.00527.x ◽

1999 ◽

Vol 42 (11-12) ◽

pp. 609-614 ◽

Cited By ~ 20

Author(s):

R. B. Caligiorne ◽

M. A. de Resende ◽

E. Dias-Neto ◽

S. C. Oliveira ◽

V. Azevedo

Keyword(s):

Polymerase Chain Reaction ◽

Restriction Fragment Length Polymorphism ◽

Gene Polymorphism ◽

Ribosomal Dna ◽

Length Polymorphism ◽

Fungal Pathogens ◽

Fragment Length Polymorphism ◽

Chain Reaction ◽

Polymerase Chain ◽

Restriction Fragment Length

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Detection of Gaeumannomyces graminis Varieties Using Polymerase Chain Reaction with Variety-Specific Primers

Plant Disease ◽

10.1094/pdis.2000.84.9.947 ◽

2000 ◽

Vol 84 (9) ◽

pp. 947-951 ◽

Cited By ~ 15

Author(s):

H. M. Fouly ◽

H. T. Wilkinson

Keyword(s):

Polymerase Chain Reaction ◽

Fungal Pathogens ◽

Gaeumannomyces Graminis ◽

Universal Primers ◽

Middle Region ◽

Chain Reaction ◽

Specific Primers ◽

Grass Roots ◽

Polymerase Chain ◽

Take All

The polymerase chain reaction (PCR) was used for detection of Gaeumannomyces graminis, the causal agent of take-all disease in wheat, oats, and turfgrass. NS5 and NS6 universal primers amplified the middle region of 18S ribosomal DNA of Gaeumannomyces species and varieties. Primers GGT-RP (5′ TGCAATGGCTTCGTGAA 3′) and GGA-RP (5′ TTTGTGTGTGAC CATAC 3′) were developed by sequence analysis of cloned NS5-NS6 fragments. The primer pair NS5:GGT-RP amplified a single 410-bp fragment from isolates of G. graminis var. tritici, a single 300-bp fragment from isolates of G. graminis var. avenae, and no amplification products from isolates of G. graminis var. graminis or other species of Gaeumannomyces. The primer pair NS5:GGA-RP amplified a single 400-bp fragment from isolates of varieties tritici and avenae. Two sets of primer pairs (NS5:GGT-RP and NS5:GGA-RP) were used in PCR reactions to detect and identify the varieties tritici and avenae either colonizing wheat, oats, or grass roots, or in culture. No amplification products were observed using DNA extracted from plants infected with eight other soilborne fungal pathogens or from uninoculated plants.

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Quantitative Multiplexed Detection of Common Pulmonary Fungal Pathogens by Labeled Primer Polymerase Chain Reaction

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2013-0592-oa ◽

2014 ◽

Vol 138 (11) ◽

pp. 1474-1480 ◽

Cited By ~ 8

Author(s):

Zhengming Gu ◽

Daelynn R. Buelow ◽

Ruta Petraitiene ◽

Vidmantas Petraitis ◽

Thomas J. Walsh ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Fungal Pathogens ◽

Bronchoalveolar Lavage Fluid ◽

Fungal Infections ◽

Lavage Fluid ◽

Quantitative Detection ◽

Target Sequence ◽

Chain Reaction ◽

Polymerase Chain ◽

Primer Polymerase Chain Reaction

Context Invasive fungal infections are an important cause of morbidity and mortality among immunocompromised patients. Objective To design and evaluate a multiplexed assay aimed at quantitative detection and differentiation of the 5 molds that are most commonly responsible for pulmonary infections. Design Using labeled primer polymerase chain reaction chemistry, an assay was designed to target the 5.8S and 28S ribosomal RNA genes of Aspergillus spp, Fusarium spp, Scedosporium spp, and members of the order Mucorales (Rhizopus oryzae, Rhizopus microsporus, Cunninghamella bertholletiae, Mucor circinelloides, Lichtheimia corymbifera, and Rhizomucor pusillus). This assay was split into 2 multiplexed reactions and was evaluated using both samples seeded with purified nucleic acid from 42 well-characterized clinical fungal isolates and 105 archived samples (47 blood [45%], 42 bronchoalveolar lavage fluid [40%], and 16 tissue [15%]) collected from rabbit models of invasive pulmonary fungal infections. Results Assay detection sensitivity was less than 25 copies of the target sequence per reaction for Aspergillus spp, 5 copies for Fusarium spp and Scedosporium spp, and 10 copies for the Mucorales. The assay showed quantitative linearity from 5 × 101 to 5 × 105 copies of target sequence per reaction. Sensitivities and specificities for bronchoalveolar lavage fluid, tissue, and blood samples were 0.86 and 0.99, 0.60 and 1.00, and 0.46 and 1.00, respectively. Conclusions Labeled primer polymerase chain reaction permits rapid, quantitative detection and differentiation of common agents of invasive fungal infection. The assay described herein shows promise for clinical implementation that may have a significant effect on the rapid diagnosis and treatment of patients' severe infections caused by these pulmonary fungal pathogens.

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Specificities of multi-primer polymerase chain reaction optimization for the detection of infectious pneumonia agents in human

Тонкие химические технологии ◽

10.32362/2410-6593-2021-16-3-225-231 ◽

2021 ◽

Vol 16 (3) ◽

pp. 225-231

Author(s):

E. S. Klochikhina ◽

V. E. Shershov ◽

V. E. Kuznetsova ◽

S. A. Lapa ◽

A. V. Chudinov

Keyword(s):

Polymerase Chain Reaction ◽

Legionella Pneumophila ◽

Fungal Pathogens ◽

Solid Phase ◽

Simultaneous Detection ◽

Clinical Diagnostics ◽

Chain Reaction ◽

Polymerase Chain ◽

Clinically Significant ◽

Primer Polymerase Chain Reaction

Objectives. The objectives of this work are the development of a multi-primer system based on the polymerase chain reaction (PCR) aimed at the simultaneous detection of six bacterial pathogens that cause human pneumonia and the determination of the parameters important for the optimization of this multi-primer system, including solid-phase PCR systems (biological microarrays).Methods. To determine the optimal parameters of the system, PCR methods were used in monoplex and multiplex formats.Results. Primers for Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenza, Legionella pneumophila, Klebsiella pneumoniae, and Streptococcus pneumoniae detection were designed, and the PCR cycling conditions were optimized. The patterns of primer design for solidphase PCR were revealed.Conclusions. The developed prototype of a system specifically identifies six clinically significant bacterial pathogens. It could be expanded for the analysis of viral and fungal pathogens and used in clinical diagnostics. A prototype of a system for pathogenic agent detection in the immobilized phase (biological microarray) was created.

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Identification of pathogens causing invasive fungal rhinosinusitis in surgical biopsies using polymerase chain reaction

The Journal of Laryngology & Otology ◽

10.1017/s0022215120001395 ◽

2020 ◽

Vol 134 (7) ◽

pp. 632-635 ◽

Cited By ~ 1

Author(s):

S Chaturantabut ◽

N Kitkumthorn ◽

A Mutirangura ◽

N Praditphol ◽

A Chindamporn ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Fungal Pathogens ◽

High Morbidity ◽

Reaction Products ◽

Chain Reaction ◽

Fresh Tissue ◽

Chain Reactions ◽

Fungal Rhinosinusitis ◽

Polymerase Chain ◽

Invasive Fungal Rhinosinusitis

AbstractBackgroundInvasive fungal rhinosinusitis is associated with high morbidity and mortality. Rapid pathogen identification is mandatory, but fresh tissue is not always available. A polymerase chain reaction method was designed in order to detect fungi in formalin-fixed paraffin-embedded samples. This was applied to a retrospective series of tissue biopsies from Thai patients with invasive fungal rhinosinusitis.MethodsTissue blocks from 64 cases yielded adequate DNA. Three sequential polymerase chain reaction were performed: ZP3 (housekeeping gene) and panfungal polymerase chain reactions, and a differentiating polymerase chain reaction based on the 5.8s ribosomal RNA and internal transcribed spacer 2 regions. The polymerase chain reaction products were then sequenced.ResultsPolymerase chain reaction identified a fungal pathogen in 20 of 64 cases (31 per cent). Aspergillus species was the most common cause of invasive fungal rhinosinusitis (nine cases). Other causes included candida (n = 4), cladosporium (n = 4), mucor (n = 1), alternaria (n = 1) and dendryphiella (n = 1) species.ConclusionPolymerase chain reaction can provide rapid identification of fungal pathogens in paraffin-embedded tissue, enabling prompt treatment of invasive fungal rhinosinusitis.

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