Optimization of DNA Extraction by Microwave Treatment and PCR Detection of Spoilage Alicyclobacillus acidoterrestris in Apple Juice

Abstract

Abstract: A simple and rapid method for microwave-assisted DNA extraction from A. acidoterrestris in juice sample was developed in this study. A pair of primers was designed based on the gene sequence encoding squalene epoxidase from Alicyclobacillus acidoterrestris published in the Genbank database to establish a PCR assay for detecting Alicyclobacillus acidoterrestris in apple juice. The specificity and sensitivity of the method was investigated by DNA amplification from 11 Alicyclobacillus spp. and 7 other bacteria using the designed specific primers. The detection limit was determined using serially diluted extracts. The results of gel electrophoresis indicated that the DNA bands of microwave-assisted samples were the same as those obtained from ZR Genomic DNA Kit, implying effective bacterial cell lysis in juice samples. The limit of detection of this method was 200 CFU/mL. Only 2 hours were required to detect A. acidoterrestris in juice sample by this method. In conclusion, the microwave-assisted DNA extraction coupled to PCR detection method provides an effective and stable approach for the online detection of Alicyclobacillus spp. from apple juice.

Key words: Alicyclobacillus acidoterrestris, apple juice, microwave treatment, polymerase chain reaction (PCR), limit of detection

CLC Number:

Q939.5

References

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