发酵饲料中乳酸菌含量检测的EMA-qPCR方法

doi:10.11843/j.issn.0366-6964.2019.10.024

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (10): 2166-2170.doi: 10.11843/j.issn.0366-6964.2019.10.024

发酵饲料中乳酸菌含量检测的EMA-qPCR方法

胡会龙¹, 魏小兵¹, 王秋霞¹, 欧长波¹, 闫艺婷², 吕玉琼³, 刘兴友^3*

1. 河南科技学院动物科技学院, 新乡 453003;
2. 畜禽智能化清洁生产河南省工程实验室, 新乡 453003;
3. 新乡学院, 新乡 453003

收稿日期:2019-03-21 出版日期:2019-10-23 发布日期:2019-10-23
通讯作者: 刘兴友,主要从事动物分子病毒学研究,E-mail:lxingyou@souhu.com
作者简介:胡会龙(1992-),河南郑州人,硕士生,主要从事动物分子病毒学研究,E-mail:707023033@qq.com;魏小兵(1988-),河南内乡人,硕士生,主要从事动物分子病毒学研究,E-mail:296603703@qq.com。胡会龙和魏小兵为同等贡献作者
基金资助:
河南省现代农业产业技术体系生猪产业技术创新团队（S2012-06-G02）；国家重点研发计划（2016YFD0500702）；河南省科技开放合作项目（182106000043）

EMA-qPCR Method for Detection of Lactic Acid Bacteria in Fermented Feed

HU Huilong¹, WEI Xiaobing¹, WANG Qiuxia¹, OU Changbo¹, YAN Yiting², Lü Yuqiong³, LIU Xingyou^3*

1. College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, China;
2. Henan Higher Education Discipline Intelligent Cleaner Production of Livestock, Xinxiang 453003, China;
3. Xinxiang University, Xinxiang 453003, China

Received:2019-03-21 Online:2019-10-23 Published:2019-10-23

摘要/Abstract

摘要： 旨在快速检测发酵饲料中乳酸菌含量，将叠氮溴乙锭（EMA）与q-PCR技术相结合，通过对诸如EMA浓度、曝光时间等影响EMA的因素进行探索，确定EMA的作用条件。在试验条件下用EMA处理已知浓度的乳酸菌菌液，提取乳酸菌DNA，结合q-PCR建立Ct值与乳酸菌含量对数值之间的标准曲线。利用标准曲线，快速计算发酵饲料中的乳酸菌含量。结果表明EMA浓度在3~4 μg·mL^-1曝光10 min可有效抑制死菌DNA的扩增，从而消除死菌DNA扩增对Ct值的影响。通过对8份发酵饲料进行检测，其结果与平板计数具有较高的符合度。EMA-qPCR方法可用于快速准确地检测发酵饲料中活的乳酸菌含量。

Abstract: To quickly detect the content of lactic acid bacteria in fermented feed, a new method was established by combining ethidium bromide monoazide(EMA) with q-PCR technology, and the factors affecting the effects of EMA, such as the EMA concentration, the action time and the illumination time, were investigated in this experiment. The known number of lactic acid bacteria was treated with EMA under the experimental conditions and then the bacterial DNA was extracted. Finally, the standard curve between the Ct value and the number of lactic acid bacteria was established following q-PCR method. The unknown number of lactic acid bacteria can be calculated by replacing the Ct value into the standard curve equation. The results showed that EMA exposure at 3-4 μg·mL^-1 concentration for 10 minutes could effectively inhibit the DNA amplification of the dead bacteria and eliminate the influence of DNA amplification on Ct value. By measuring 8 parts of fermented feed, the results have a high degree of conformity with the plate count. Therefore, it is feasible to quickly and accurately detect the quantity of viable lactic acid bacteria in fermented feed with EMA-qPCR.

中图分类号:

S816.601.7

胡会龙, 魏小兵, 王秋霞, 欧长波, 闫艺婷, 吕玉琼, 刘兴友. 发酵饲料中乳酸菌含量检测的EMA-qPCR方法[J]. 畜牧兽医学报, 2019, 50(10): 2166-2170.

HU Huilong, WEI Xiaobing, WANG Qiuxia, OU Changbo, YAN Yiting, Lü Yuqiong, LIU Xingyou. EMA-qPCR Method for Detection of Lactic Acid Bacteria in Fermented Feed[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(10): 2166-2170.

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发酵饲料中乳酸菌含量检测的EMA-qPCR方法

EMA-qPCR Method for Detection of Lactic Acid Bacteria in Fermented Feed

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