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 × 100 cfu/ml for the test sample compared with a detection limit of 1.6 × 103 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.
Highlights
Polymerase chain reaction (PCR) is performed after DNA isolation
Because a low detection limit has never been obtained for prokaryotic cells through intermediate direct qPCR using crude or classical approaches involving preventive DNA isolation[12,13,14], here we report the detailed outcomes of the present study
To evaluate the disruption rate of C. muytjensii cells during polymerase chain reaction (PCR) thermal cycling, PCR elongation was performed using a direct qPCR master mix for the pellet and supernatant obtained from C. muytjensii previously subjected to 50 rounds of PCR thermal cycling in physiological saline or direct qPCR components (Table 1)
Summary
Real-time PCR (qPCR), known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA 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. ® qPCR has been performed by using commercially available kits, such as MightyAmp DNA Polymerase Ver.[2], to suppress the function of PCR inhibitors in experimental samples (Takara-Bio, Ohtsu, Japan)[11] The goal of this experiment was to conduct simple and rapid genomic research and routine food/environmental/clinical testing to detect DNA from nearly a single prokaryotic cell with solid cell walls. MO, USA), to adsorb PCR inhibitors from the test samples, a non-ionic detergent to modify the structures and dissolve the proteins of bacterial cell walls, and a calcium chelating agent[18,19,20,21]
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