scholarly journals Development of Nested Polymerase Chain Reaction Detection of Mycosphaerella spp. and Its Application to the Study of Leaf Disease in Eucalyptus Plantations

2007 ◽  
Vol 97 (2) ◽  
pp. 132-144 ◽  
Author(s):  
M. Glen ◽  
A. H. Smith ◽  
S. R. H. Langrell ◽  
C. L. Mohammed
Keyword(s):  
Polymerase Chain Reaction ◽  
Sequence Similarity ◽  
Nuclear Ribosomal Dna ◽  
Chain Reaction ◽  
Specific Primers ◽  
Nested Polymerase Chain Reaction ◽  
Double Blind ◽  
Leaf Disease ◽  
Eucalyptus Plantations ◽  
Polymerase Chain

Mycosphaerella leaf disease (MLD) is a serious disease of two of the major eucalypt species grown in temperate regions worldwide, Eucalyptus globulus and E. nitens. More than 30 species of Mycosphaerella have been reported on eucalypts worldwide. Accurate, rapid, and early discrimination of Mycosphaerella spp. causing crown damage to E. globulus and E. nitens will assist the development of sustainable management strategies. This study describes the development, and incorporation in a nested polymerase chain reaction (PCR) approach, of specific primers for the detection and identification of Mycosphaerella spp. commonly reported from leaf lesions of E. globulus and E. nitens in Australia. Primer design was assisted by sequence alignment and phylogenetic analysis of 165 nonredundant sequences from the nuclear ribosomal DNA internal transcribed spacer regions of Mycosphaerella and related species. Phylo-genetic analysis revealed very high sequence similarity for two taxon groups, Mycosphaerella grandis and M. parva, and M. vespa, M. ambi phylla, and M. molleriana, and primers were designed to differentiate each of the two groups. Three other species, M. cryptica, M. nubilosa, and M. tasmaniensis, were distinct and distinguished by species-specific primers. In double-blind trials, the detection test accurately and rapidly identified Mycosphaerella spp. in cultures and discriminated against other pathogens that co-occur in or on Eucalyptus leaves, thereby verifying its reliability. The detection test has an internal amplification control in the first-round PCR with fungal-specific primers to raise confidence in test results, particularly to highlight negative results due to PCR inhibition. When applied to DNA extracted from leaf or stem samples either as multiple or single lesions, it detected and identified up to five Mycosphaerella spp. or taxon groups in both positively identified and in young (putative) MLD lesions. The samples were 20 mm2 or larger in surface area and were collected while undertaking disease rating assessments in an experimental investigation of Eucalyptus plantations and regrowth forest. Using nested PCR detection, Mycosphaerella spp. were positively identified in 2 days, 1 to 5 months earlier than by classical methods, demonstrating the potential application of this detection test to the early discrimination of MLD components in ecological, epidemiological, and genetic investigations.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

Detection of hepatitis “C” virus in formalin-fixed liver tissue by nested polymerase chain reaction

1992 ◽  
Vol 37 (4) ◽  
pp. 310-314 ◽  
Author(s):  
Richard Sallie ◽  
Anne Rayner ◽  
Bernard Portmann ◽  
A. L. W. F. Eddleston ◽  
Roger Williams
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Liver Tissue ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Formalin Fixed

The fate of a genetically modifiedPseudomonasstrain and its transgene during the composting of poultry manure

2004 ◽  
Vol 50 (6) ◽  
pp. 415-421 ◽  
Author(s):  
J Guan ◽  
J L Spencer ◽  
M Sampath ◽  
J Devenish
Keyword(s):  
Polymerase Chain Reaction ◽  
Incubation Temperature ◽  
Genetically Modified ◽  
Chicken Manure ◽  
Detection Sensitivity ◽  
Plate Count ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Soil Microcosms ◽  
Polymerase Chain

The fate of the genetically modified (GM) Pseudomonas chlororaphis strain 3732 RN-L11 and its transgene (lacZ insert) during composting of chicken manure was studied using plate count and nested polymerase chain reaction (PCR) methods. The detection sensitivity of the nested PCR method was 165 copies of the modified gene per gram of moist compost or soil. Compost microcosms consisted of a 100-g mixture of chicken manure and peat, whereas soil microcosms were 100-g samples of sandy clay loam. Each microcosm was inoculated with 4 × 1010CFU of P. chlororaphis RN-L11. In controlled temperature studies, neither P. chlororaphis RN-L11 nor its transgene could be detected in compost microcosms after incubation temperature was elevated to 45 °C or above for one or more days. In contrast, in the compost microcosms incubated at 23 °C, the target organism was not detected by the plate count method after 6 days, but its transgene was detectable for at least 45 days. In compost bins, the target organism was not recovered from compost microcosms or soil microcosms at different levels in the bins for 29 days. However, the transgene was detected in 8 of the 9 soil microcosms and in only 1 of the 9 compost microcosms. The compost microcosm in which transgene was detected was at the lower level of the bin where temperatures remained below 45 °C. The findings indicated that composting of organic wastes could be used to reduce or degrade heat sensitive GM microorganisms and their transgenes.Key words: composting, genetically modified Pseudomonas strain, transgene, polymerase chain reaction.

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