New Developments in PCR

André, Chas P.

doi:10.2144/000113149

Cited by 3 publications

(1 citation statement)

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“…This polymerase is reported to lead to a 25-fold lower error rate as compared to common Thermus aquaticus Brock & Freeze ( Taq ) polymerase (Wang et al 2004 ). Besides a huge increase in efficiency, fusion polymerases are resistant to various PCR inhibitors and have faster overall performance (André 2009 ). This technology opens possibilities to develop rapid diagnostic methods (e.g., haplotype and species determination) for plant pathogenic fungi by omitting the DNA purification step.…”

Section: Introductionmentioning

confidence: 99%

Application of direct PCR in rapid rDNA ITS haplotype determination of the hyperparasitic fungus Sphaeropsis visci (Botryosphaeriaceae)

et al. 2014

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BackgroundThe plant pathogenic fungus, Sphaeropsis visci a dark-spored species of Botryosphaeriaceae, which causes the leaf spot disease of the European mistletoe (Viscum album). This species seems to have potential as a tool for biological control of the hemiparasite. For the rapid detection of S. visci haplotypes we tested a direct PCR assay without prior DNA purification. This approach was based on a polymerase enzyme from the crenarchaeon Sulfolobus solfataricus engineered by fusion protein technology, which linked the polymerase domain to a sequence non-specific DNA binding protein (Sso7d).FindingsMost isolates of Sphaeropsis visci grouped together in our phylogenetic analyses, indicating that isolates had a previously reported haplotype sequence, which is commonly found in the analyzed Hungarian population. This haplotype was also reported from diseased mistletoe bushes from other European countries. We further identified unique single nucleotide polymorphisms (SNPs) in the ITS region, which were specific to the only well resolved clade in the phylogenetic analysis.ConclusionsThe diPCR approach allowed amplification of ITS rRNA gene directly from small amounts of fungal samples without prior DNA extraction. This simple bioassay in plant disease management enables collection of genomic data from fungal plant pathogen populations.

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Section: Introductionmentioning

confidence: 99%