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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2017, Vol. 43 Issue (2): 153-162    DOI: 10.3785/j.issn.1008-9209.2016.04.11
生物科学与技术     
检测乙酰微小杆菌的双重实时荧光定量聚合酶链式反应方法的建立
 高灿灿，刘佳玫，栗军杰，陆兆新，吕凤霞，张充，赵海珍，别小妹*
南京农业大学食品科学技术学院，南京210095
 Identification of Exiguobacterium acetylicum by a double real- time polymerase chain reaction assay
GAO Cancan, LIU Jiamei, LI Junjie, LU Zhaoxin, Lü Fengxia, ZHANG Chong, ZHAO Haizhen, BIE Xiaomei*
（College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China）
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摘要：  以鲜切叶菜中的乙酰微小杆菌（Exiguobacterium acetylicum）为研究对象，建立定量检测E. acetylicum 的双重实时荧光定量聚合酶链式反应（real-time polymerase chain reaction, RT-PCR）方法。以前期试验发掘到的微小杆菌（Exiguobacterium sp.）的特异性基因P401_RS0117025 和E. acetylicum 的特异性基因oxi_50582462 为检测靶点，对其设计特异性探针，并对反应体系进行优化，以12株E. acetylicum 为阳性对照，以4株微生菌属内其他种的菌株以及10株非微小杆菌的腐败菌/致病菌作为阴性对照，对建立的RT-PCR进行特异性检测；通过标准曲线的制备、灵敏度、可重复性来评价该方法的有效性。最后，应用该RT-PCR方法对不同贮藏日期鲜切叶菜中的E. acetylicum 进行检测。结果表明：本研究设计的探针特异性良好；检测靶点P401_RS0117025 与oxi_50582462 的标准曲线的R2值分别为0.994和0.999，且重复性好；该方法的DNA检测限为1.629×10－7 ng/μL，纯菌菌落检测限为3.4 CFU/mL，并能对鲜切叶菜中的E. acetylicum 进行灵敏检测。此外，还建立了“Ct - Nt（菌体浓度）”的数量关系，能对供试样品中的E. acetylicum 进行绝对定量。总之，本研究建立了准确、灵敏、高效检测食品中腐败菌E. acetylicum 的RT-PCR定量方法，为食品货架期预测提供了理论依据。
Abstract: Exiguobacterium sp. has been figured out to be the main spoilage bacteria on many kinds of food, and it has the potential to be the main spoiler. Exiguobacterium has already been isolated from extreme environments and they have evolved and acquired some resistance including thermotolerance, cold resistance, alkali resistance, and osmotolerance. The Exiguobacterium can normally metabolize under 4 ℃ storage temperature. Further, Exiguobacterium has strong proteolysis ability to damage nutrient and food, and it can form biofilm, not only increasing the resistance to detergents and preservatives, but also consuming nutrients to grow and reproduce. The present study aimed to establish a rapid identification method of Exiguobacterium acetylicum based on a double real-time polymerase chain reaction (RT-PCR). The study took the genes P401_RS0117025 and oxi_50582462, which were screened in the early experiment and were specific to Exiguobacterium sp. and E. acetylicum respectively, as detection targets. The specificity of the PCR assay was verified with the DNA of 12 E. acetylicum strains and 14 non-E. acetylicum strains; the effectiveness of the assay was evaluated by the foundation of standard curve, as well as its repeatability. The sensitivity of different DNA and cell concentrations was identified. The results demonstrated that the specific genes and primers of the early experiment were both well-specific. The specific evaluation experiment results showed that the probes T-291 and T-2B were also well-specific. The R2 values of standard curve were 0.994 and 0.999, respectively, indicating that the assay was credible, and the RT- PCR showed high repeatability. When DNA of different concentrations were added as templates, there was no obvious different in standard deviation of threshold (Ct), and the coefficient of variation of the same concentration was fluctuation during 0.51 to 1.12. Even though at 1.629×10－4 ng/μL, its repeatability was well maintained, which indicated that the assay could showed high repeatability at low DNA concentration. The sensitivity-evaluation showed that the DNA detection limit of the assay was 1.629×10－7 ng/μL, and the detection limit of pure bacteria colonies was 3.4 CFU/mL without enrichment culture. In addition, the high-sensitivity resulted in its outcoming of short- time cast, for it did not need the step of enrichment culture, which was different from other studies. Finally, the mathematical relationship between Ct and Nt colony-forming units (CFU) was developed by software Origin 9. It was Ct=－1.759× Nt +31.678, the R2 value of which was 0.939. When applied to the fresh-cut leafy vegetables, the assay and the equation could detect rapidly and accurately the CFU on them. In sum, this study establish as an accurate, sensitive and efficient double RT- PCR assay to detect quantitatively food spoilage bacteria E. acetylicum. It lays a theoretical basis for food shelf- life prediction, and provides some references to the supply chain of fresh-cut vegetables.
出版日期: 2017-03-25
CLC:  Q-31  
基金资助:  国家自然科学基金（31271828）
通讯作者: 别小妹(http://orcid.org/0000-0002-2175-0164)，E-mail: bxm43@njau.edu.cn   
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引用本文:

高灿灿，刘佳玫，栗军杰，陆兆新，吕凤霞，张充，赵海珍，别小妹. 检测乙酰微小杆菌的双重实时荧光定量聚合酶链式反应方法的建立[J]. 浙江大学学报(农业与生命科学版), 2017, 43(2): 153-162.

GAO Cancan, LIU Jiamei, LI Junjie, LU Zhaoxin, Lü Fengxia, ZHANG Chong, ZHAO Haizhen, BIE Xiaomei.  Identification of Exiguobacterium acetylicum by a double real- time polymerase chain reaction assay. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(2): 153-162.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2016.04.11    或    http://www.zjujournals.com/agr/CN/Y2017/V43/I2/153

 
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