Study On The Application Of DHPLC Technique In Rapid Detection Of Food Microorganism

In this paper, based on the molecular biology technology, The flatoxigenic bacteria ofAspergillus, candida albicans, Clostridium botulinum-A，Bacillus coagulans,Lactobacillus fermenti were selected to establish a rapid detection method by denaturinghigh performance liquid chromatography (DHPLC) and polymerase chain reaction (PCR).Our aim is to offer theoretical basis and scientific support for quick testing microorganismsin food. The main results are as follows after investigation:1. The technologies of PCR and DHPLC were applied to provide a method fordetection flatoxigenic bacteria in food rapidly. AflR gene from a coded aflatoxinbiosynthesis was taken as the target design primer. The PCR system causing aflatoxincould rapidly be recognized by the method established and optimized. The amplificationsegment was184bp. The result showed that specification and sensitivity were better. Theminimum detection limit could reach100cfu/ml. The75samples from different food wereanalyzed and identified two positive samples, those results were consistent with those ofold methods and PCR applied.2. The technologies of PCR and DHPLC were applied to provide a method forcandida albicans detected in food rapidly. The unique target sequence acid protease geneSAP6from candida albicans was selected as the specific design primer, that is, the primerwere respectively F-5`-CTGGGTCTTCTGATTTGTGG-3`and R-5`-CTGGTAGCTTCGTTGGTTTG-3`, and the amplification segment was307bp. PCR systemwas established and optimized. Our results indicated that the specification was better, andthe limit of sensitivity reached1.0×103cfu/ml. The candida albicans in milk powder couldbe rapidly and truly determined.3. The technologies of PCR and DHPLC were applied to provide a method forclostridium botulinum-A determined in food rapidly. The A type-botulinum neurotoxingene from clostridium botulinum-A was selected as the specific primer. Twenty-threestrains from non-clostridium botulinum-A and clostridium perfringens were considered asa control for specific analysis, and the DNA template was diluted into different gradient toanalyze the sensitivity. The result showed that the primer had a better specification andsensitivity, and lowest detectable limit could achieve for110ng/tube.4. Primer was designed by using specificity gene Poly-6-1glycosidase enzymes fromBacillus coagulans and fermentation lactic acid extensions factor (EF-Tu) as target gene: (1) Condensation bacillus primer sequence: F-5’-TACGGCATTGGCAAGTATCA-3’, R-CGACATGATTTGGTTTTCCA-3’, amplification555bp;(2) fermentationlactobacillus primer sequence: F-5’-TGATGGTCCTATGCCACAAA-3’, R-TCAACACCGGTAACAGTGGA-3’, amplification segment was456bp. After establish andoptimize the PCR system, the specification was better. Condensation bacillus sensitivitydetection limit was1.0×102cfu/ml, and lactobacillus fermentation was7.6×102cfu/ml.