Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates

The fast and reliable detection of bacterial spores is of great importance and still remains a challenge. Here we describe a direct RNA-based diagnostic method for the specific detection of viable bacterial spores which does not depends on an enzymatic amplification step and therefore is directly appropriate for quantification. The procedure includes the following steps: (i) heat activation of spores, (ii) germination and enrichment cultivation, (iii) cell lysis, and (iv) analysis of 16S rRNA in crude cell lysates using a sandwich hybridization assay. The sensitivity of the method is dependent on the cultivation time and the detection limit; it is possible to detect 10 spores per ml when the RNA analysis is performed after 6 h of enrichment cultivation. At spore concentrations above 106 spores per ml the cultivation time can be shortened to 30 min. Total analysis times are in the range of 2–8 h depending on the spore concentration in samples. The developed procedure is optimized at the example of Bacillus subtilis spores but should be applicable to other organisms. The new method can easily be modified for other target RNAs and is suitable for specific detection of spores from known groups of organisms.

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“…The procedure was performed as described by Rautio et al (2003) and Thieme et al (2008) with small modifications.…”

Section: Methodsmentioning

confidence: 99%

“…Sandwich hybridization assays (SHA) are suitable for a rapid and quantitative RNA detection (Rautio et al, 2003 ). These methods are based on the hybridization of a target RNA (or denatured DNA) with two specific oligonucleotide probes.…”

Section: Introductionmentioning

confidence: 99%

Quantitative and sensitive RNA based detection of Bacillus spores

et al. 2014

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“…Thus two hybridization events are involved. In the first event the immobilized capture probe binds to the target sequence binds event, and in the second hybridization event, a signal probe linked to a recorder molecule [ 47 , 48 ], such as a fluorochrome or digoxigenin (DIG) binds to the first complex to facilitate its detection. To detect the target species, only one of the two probes need be specific.…”

Section: Molecular—cell-free Formatmentioning

confidence: 99%

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species

Medlin

Orozco

2017

Sensors

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Molecular techniques to detect organisms in aquatic ecosystems are being gradually considered as an attractive alternative to standard laboratory methods. They offer faster and more accurate means of detecting and monitoring species, with respect to their traditional homologues based on culture and microscopic counting. Molecular techniques are particularly attractive when multiple species need to be detected and/or are in very low abundance. This paper reviews molecular techniques based on whole cells, such as microscope-based enumeration and Fluorescence In-Situ Hybridization (FISH) and molecular cell-free formats, such as sandwich hybridization assay (SHA), biosensors, microarrays, quantitative polymerase chain reaction (qPCR) and real time PCR (RT-PCR). Those that combine one or several laboratory functions into a single integrated system (lab-on-a-chip) and techniques that generate a much higher throughput data, such as next-generation systems (NGS), were also reviewed. We also included some other approaches that enhance the performance of molecular techniques. For instance, nano-bioengineered probes and platforms, pre-concentration and magnetic separation systems, and solid-phase hybridization offer highly pre-concentration capabilities. Isothermal amplification and hybridization chain reaction (HCR) improve hybridization and amplification techniques. Finally, we presented a study case of field remote sensing of harmful algal blooms (HABs), the only example of real time monitoring, and close the discussion with future directions and concluding remarks.

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“…Detection is possible only when both probes are hybridized to the target molecule. Unlike PCR, in which the enzyme‐based amplification is affected by the presence of PCR inhibitors, in SHA, there is no amplification, and therefore, the hybridization of the probes can be performed on crude cellular extracts , without lengthy and laborious DNA/RNA extraction and purification steps. Because it shares a similar target identification principle, SHA provides comparable levels of specificity to PCR.…”

Section: Introductionmentioning

confidence: 99%

“…However, these sensors are complicated, costly and require long hybridization times (up to 9 hours) . Therefore, the majority of the widely implemented detection systems are optically‐based and are assisted by low‐cost enzyme‐based signal amplification methods . While this approach is faster (2‐4 hours) than electrochemical sensing, it is approximately five orders of magnitude less sensitive (LoD of 11 pM) .…”

Section: Introductionmentioning

confidence: 99%

Detecting nucleic acid fragments in serum using a magnetically modulated sandwich assay

Margulis

Ashri

Cohen

et al. 2019

Journal of Biophotonics

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We present a novel assay for rapid and highly sensitive detection of specific nucleic acid fragments in human serum. In a magnetic modulation biosensing (MMB) system, magnetic beads and fluorescently labeled probes are attached to the target analyte and form a "sandwich" complex.An alternating external magnetic field gradient condenses the magnetic beads (and hence the target molecules with the fluorescently labeled probes) to the detection volume and sets them in a periodic motion, in and out of a laser beam. A synchronous detection enables the removal of background signal from the oscillating target signal without complicated sample preparation. The high sensitivity of the MMB system, combined with the specificity of a sandwich hybridization assay, enables detection of DNA fragments without enzymatic signal amplification. Here, we demonstrate the sensitivity of the assay by directly detecting the EML4-ALK oncogenic translocation sequence spiked in human serum. The calculated limit of detection is 1.4 pM, which is approximately 150 times better than a conventional plate reader. In general, the MMB-assisted SHA can be implemented in many other applications for which enzymatic amplification, such as PCR, is not applicable and where rapid detection of specific nucleic acid targets is required.

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Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates

Cited by 56 publications

References 31 publications

Quantitative and sensitive RNA based detection of Bacillus spores

Quantitative and sensitive RNA based detection of Bacillus spores

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species

Detecting nucleic acid fragments in serum using a magnetically modulated sandwich assay

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