Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay

Abstract

Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, backgroundfree ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5 and 3 ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 10 4 . A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.

Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays

“…A straightforward approach to detect PCR inhibition is the inclusion of an internal amplification control (IAC) (6,7,16,28). An IAC is a nontarget DNA sequence that is coamplified with the target under the same reaction conditions and in the same reaction tube.…”

Alien fungal species on asymptomatic live woody plant material imported into Canada

“…It also circumvents the limitations of DNA probe-based methods, which generally deal only with known and well-characterized organisms with available sequence data (Szemes et al 2005;Van Doorn et al 2009). …”

Emerging Molecular Assays for Detection and Characterization of Respiratory Viruses

“…119 Besides viral pathogens, padlock probe-based techniques have been rapidly extended in recent years to the identification and characterization of bacterial and fungal pathogens. [120][121][122][123][124] In addition to the applicability of padlock probes for direct target detection, a universal primer binding site can be introduced into the probe and used for MLPA (see previous discussion).…”