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首页> 中文期刊> 《农业工程学报》 >原料奶中微生物区系ERIC-PCR DNA指纹图谱的建立

原料奶中微生物区系ERIC-PCR DNA指纹图谱的建立

         
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摘要

Enterobacterial Repetitive Intergenic Consensus (ERIC) primarily exists in enterobacteria. The sequence length is about 124-127 bp, which contain about 44 bp highly conservative core sequences in its center. ERIC-PCR was widely applied to research the intestinal microbial community composition and diversity. However, we rarely saw domestic and foreign related reports about the ERIC-PCR method which was adopted to analyze the microbial community compositon in raw milk. ERIC-PCR molecular biotechnology was used to research the microbial community, and DNA fingerprints on microbes in raw milk in North China and in Central South China was established in this paper. First, the bacteria DNA extraction method in raw milk had to be built. This paper utilized the CTAB extraction method. Secondly, we needed to determine the optimal ERIC-PCR reaction condition. In order to obtain a distinct and high quality fingerprint, influencing factors on the ERIC-PCR system for amplification was optimized, which included annealing temperature, Mg2+ concentration, primer concentration and Taq DNA polymerase enzymatic activity. Randomly selecting four DNA samples, each condition set four gradients. The best level of all the factors were that the optimum annealing temperature was 52 ,℃ the optimum Mg2+concentration was 2mmol/L, the optimum primer concentration was 0.24μmol/L and the optimum Taq DNA polymerase enzymatic activity was 2.5U. Thirdly, a DNA fingerprint on microbe in raw milk in North China and in Central South China had to be established. ERIC-PCR product was conducted through electrophoresis in 1.7%agarose gel, then the electrophoresis product was analyzed by a Gel Imaging System. We utilized Quantity One 4.6.2 software to analyze the ERIC-PCR DNA fingerprint. Diversity analysis of the fingerprint adopted the Shannon-Wiener index. The computational formula is H=-∑Pi lnPi (Pi=Ni/N, Ni represents the i ERIC band area under the peak of one sample and N is on behalf of all the ERIC band area under the peak of the sample.) Similarity analysis of the fingerprint was calculated automatically by the software according to a Dice coefficient, then phylogenetic tree diagram was drawn by using the weighted average of the distance (UPGMA) analytical method. By comparing DNA fingerprints of two regions, we could draw a conclusion that the microbial community structure of different raw milk samples from near areas were highly similar and had some dominant microflora respectively, while the different raw milk samples from distinct areas had lower similarity. This study indicated that the establishment of a specific DNA fingerprint in different regions could provide scientific proof for the monitoring of quality and safety in raw milk.%　　为了研究原料奶中微生物区系的结构，该研究采用肠杆菌基因间保守重复序列ERIC-PCR技术，建立了华北和中南地区原料奶中微生物区系的ERIC-PCR DNA指纹图谱。为获得清晰且高质量的图谱，对ERIC-PCR的反应条件进行了优化，确定最佳条件为：退火温度52℃、MgCl21μL、引物0.6μL、Taq酶0.5μL。对2个地区指纹图谱进行综合分析鉴定。结果显示，地理位置距离较近即相近地域原料奶样品其微生物群落的构成相似性较高，存在一定特征性优势菌群；距离较远则相似性较低。研究结果提示，建立中国不同地区原料奶微生物区系 DNA的特征图谱，将为原料奶质量与安全的监测提供参考。

著录项

  • 来源
    《农业工程学报》 |2013年第11期|261-269|共9页
  • 作者

    陈庆森; 高文茹; 朱晨晨; 阎亚丽; 庞广昌; 胡志和;

  • 作者单位

    天津市食品生物技术重点实验室;

    天津商业大学生物技术与食品科学学院;

    天津 300134;

    天津市天狮生命科学与技术研究院技术支持部;

    天津 301700;

    天津市食品生物技术重点实验室;

    天津商业大学生物技术与食品科学学院;

    天津 300134;

    天津市食品生物技术重点实验室;

    天津商业大学生物技术与食品科学学院;

    天津 300134;

    天津市食品生物技术重点实验室;

    天津商业大学生物技术与食品科学学院;

    天津 300134;

    天津市食品生物技术重点实验室;

    天津商业大学生物技术与食品科学学院;

    天津 300134;

  • 原文格式 PDF
  • 正文语种 chi
  • 中图分类 基础科学;
  • 关键词

    微生物; 优化; 农产品; 原料奶; ERIC-PCR; DNA指纹图谱;

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