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Thereafter, it was immediately chilled in an ice-water bath.
This treatment simulated high-temperature, short-time pasteurization and avoided denaturing the DNA gyrase/topoisomerase IV of bacteria.
Developing rapid PCR methods to substitute for the culture method is a pressing matter in clinical and food hygiene tests.
Most clinical samples are derived from patients administered antibiotics.
Thus, we used DNA gyrase/topoisomerase IV and mammalian topoisomerase poisons (here abbreviated as T-poisons) together with EMA Light.
Herein, we report that EMA Light randomly cleaved chromosomal DNA of heat-treated, but not live, , DNA amplification was not completely suppressed by EMA Light only.
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CIN, purchased from Fluka Chemie Gmb H (Buchs, Germany), was dissolved in physiological saline.
JCM 2873 was cultured at 30°C in brain heart infusion (BHI) broth (Eiken Kagaku, Tokyo, Japan).
To prepare live bacterial suspensions, bacteria in the logarithmic growth phase were suspended in physiological saline.
The PCR amplification of DNA from dead bacteria was inhibited by cross-linking action (23, 27-29), and the PCR signal from dead bacteria was reduced to 1/300 to 1/1,000 (23, 27, 29).
It has been reported that pasteurized milk contains 10 cells/ml of injured/dead bacteria (1, 30).
To overcome this disadvantage, reverse transcriptase PCR that targets m RNA has been used.