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Diagnostic application of padlock probes--multiplex detection of plant pathogens using universal microarrays.
- Source :
-
Nucleic acids research [Nucleic Acids Res] 2005 Apr 28; Vol. 33 (8), pp. e70. Date of Electronic Publication: 2005 Apr 28. - Publication Year :
- 2005
-
Abstract
- Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.
- Subjects :
- Animals
Fungi genetics
Fungi isolation & purification
Nematoda genetics
Nematoda isolation & purification
Oomycetes genetics
Oomycetes isolation & purification
Plant Diseases parasitology
Molecular Diagnostic Techniques
Oligonucleotide Array Sequence Analysis methods
Oligonucleotide Probes chemistry
Plant Diseases microbiology
Subjects
Details
- Language :
- English
- ISSN :
- 1362-4962
- Volume :
- 33
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Nucleic acids research
- Publication Type :
- Academic Journal
- Accession number :
- 15860767
- Full Text :
- https://doi.org/10.1093/nar/gni069