Studies On Rapid Detection Of Alicyclobacillus Acidoterrestris By PCR Method In Apple Juice Concentrate

Alicyclobacillus acidoterrestris, also called as thermophic acidophlic bacteria(TAB), is an important target micro-organism in quality control of apple juice concentrate(AJC). The spores of A. acidoterrestris are relatively heat resistant and can surive conventional pasteurisation treatment applied to AJC, and they can germinate and outgrow under suitable conditions causing spoilage. In the international trade of AJC, there are always demands for A. acidoterrestris being less than ICFU/10g AJC. A, acidoterrestris is one of the most serious quality problems in China AJC industry, for no feasible ways can control it at present.Therefore, rapid detection of such bacteria is essential for the quality control of AJC product. At present A. acidoterrestris is detected by plate count method, but it takes too much time, generally four to five days being required. Rapid and accurate detection methods are expected to develop.Polymerase chain reaction (PCR) is an elegent technique of creating copies of specific fragments of DNA, which rapidly amplifies a single DNA fragment into millions. The technology of PCR can be used to detect microorganisms quickly as long as the primers used are suitable.In this paper, two oligonucleotide primers were designed from the V2 and V4 regions of A. acidoterrestris' 16S rDNA sequence. A PCR method for rapid and specific detecting of A. acidoterrestris is investigated, and its reliability is judged as well. Diagnostic kit for detecting of TAB is developed; its performance is also assessed by comparison with routine detecting method.The results of the studies are listed as follows:1. The primers used are strictly specific. Only A. acidoterrestris produces an intense band of 294bp after amplification; to the contrast, all the other bacteria strains can't produce any bands during the same amplification.2. The optimum parameter for PCR reactionary system: Taq DNA polymerase 2U, Mg2+ 2.0mmol/L in 50L volume. And the annealing temperature is selected at 58 C. The PCR amplifying is done according to the following program: pre-denaturation at94 ℃ for 4min;35cyclesof94 ℃ for30S,58 ℃ for 30S,72℃ for 30S, final extension at72℃ for 5mhio3. The detection limit of the PCR method is Ipg total genomic DNA. And it can detect A, addoterrestris as low to 5.0+103CFU/mL.4. To detect A. addoterrestris in low concentrate, the enrichment is necessary. The optimum enrichment culture condition is : 402 medium, pH 4.0 at 45℃ for 15h.5. Three means for DNA extraction (CTAB SDS, water- boiling) are investigated. The boiling-water bath means is simple and effective, so it is choosed as the optimum DNA extracting approach.6.The complete PCR detecting system is: AJC 10g-diluted by sterilized water -filtered through membrane( b 25mm, pore size 0.22 m) - membrane translated to 402 medium-70℃ heat shock for 10 min-cultured at 45℃for 15h- centrifuge at 12000rpm for 10min-sendiment moved to microfuge-tube-DNA extracted-PCR amplifying-results analysis.The PCR detection can be done in 18-20 hours. The detecting results obtained by PCR method coincide with those of KFL's.7. The self-designed diagnose kit performs well. The kit's detecting results are reliable and repeatable. Its period of validity is at least 60 days (the maximum period of validity needs further research).