ArticleScientific reports2016
A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.
Article in Scientific reports, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed.
- Direct quantification of waterborne viruses via high-temperature and high-pressure treatment: a simplified nucleic acid extraction-free approach.Scientific reports · 2026Article
- Long-duration environmental biosensing by recording analyte detection in DNA using recombinase memory.Applied and environmental microbiology · 2024Article
- Can qPCR replace the standard plaque assay in the ASTM F1671 to assess personal protective equipment barrier performance?International journal of clothing science & technology · 2024Article
- Ultrasensitive qPCR platform for rapid detection of bacterial contamination of raw biological samples at the point of care.Heliyon · 2023Article
- Exclusive use of digital PCR allows an absolute assay of heat-killed Lactobacilli in foods targeting multiple copies of 16S rDNA.Scientific reports · 2020Article
- Separation of Biological Entities From Human Blood by Using Magnetic Nanocomposites Obtained From Zeolite Precursors.Molecules (Basel, Switzerland) · 2020Article
- Genotyping of Single Nucleotide Polymorphisms Using Allele-Specific qPCR Producing Amplicons of Small Sizes Directly from Crude Serum Isolated from Capillary Blood by a Hand-Powered Paper Centrifuge.Diagnostics (Basel, Switzerland) · 2019Article
- Innovative Use of Palladium Compounds To Selectively Detect Live Enterobacteriaceae in Milk by PCR.Applied and environmental microbiology · 2016Article
- Immuno-modulation by heat-killedInternational journal of immunopathology and pharmacologyReview
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3 authors.
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Abstract
Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.
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