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

Abstract

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.

CLC Number:

S816.601.7

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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References 18

[1]	WANG W X, ZHOU L J, GU X H, et al. Occurrence and distribution of antibiotics in surface water impacted by crab culturing:a case study of Lake Guchenghu, China[J]. Environ Sci Pollut Res, 2018, 25(23):22619-22628.
[2]	崔艳红, 韩庆功,崔艺佳,等. 益生菌复合发酵料对断奶仔猪消化环境、血清生化指标和代谢激素水平的影响[J]. 西北农业学报, 2018, 27(1):16-23.
	CUI Y H, HAN Q G, CUI Y J, et al. Effects of probiotics composite fermentation feed on gastrointestinal circumstance, serum biochemical and metabolic hormones indexes of weaning piglets[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2008, 27(1):16-23. (in Chinese)
[3]	张秀林, 魏小兵,欧长波,等. 益生菌发酵饲料对仔猪生长和免疫功能影响的研究进展[J]. 中国畜牧兽医, 2017, 44(2):476-481.
	ZHANG X L, WEI X B, OU C B, et al. Research progress of probiotics fermented feed effects on growth performance and immune function in piglets[J]. China Animal Husbandry & Veterinary Medicine, 2017, 44(2):476-481. (in Chinese)
[4]	WANG C, SHI C Y, ZHANG Y, et al. Microbiota in fermented feed and swine gut[J]. Appl Microbiol Biotechnol, 2018, 102(7):2941-2948.
[5]	RAHMAN M, BORA J R, SARMA A K, et al. Effect of deep litter housing and fermented feed on carcass characteristics and meat quality of crossbred Hampshire pigs[J]. Vet World, 2015, 8(7):881-887.
[6]	袁文华, 赵文文,袁慧坤,等. 无抗发酵饲料在动物生产上的应用研究进展[J]. 黑龙江畜牧兽医, 2018(9):59-62.
	YUAN W H, ZHAO W W, YUAN H K, et al. Research progress on the application of non-fermentable feed in animal production[J]. Heilongjiang Animal Science and Veterinary Medicine, 2018(9):59-62. (in Chinese)
[7]	谯仕彦, 侯成立,曾祥芳. 乳酸菌对猪肠道屏障功能的调节作用及其机制[J]. 动物营养学报, 2014, 26(10):3052-3063.
	QIAO S Y, HOU C L, ZENG X F. Regulation and mechanism of lactic acid bacteria on porcine intestinal barrier function[J]. Chinese Journal of Animal Nutrition, 2014, 26(10):3052-3063. (in Chinese)
[8]	CANH V D, KASUGA I, FURUMAI H, et al. Viability RT-qPCR combined with sodium Deoxycholate pre-treatment for selective quantification of infectious viruses in drinking water samples[J]. Food Environ Virol, 2019, 11(1):40-51.
[9]	FUKUZAWA S, SHIHO H, FUJITA T. Selective detection of DNA from viable Mycobacterium tuberculosis complex strains using the EMA-PCR method[J]. Jpn J Infect Dis, 2019, 72(1):19-22.
[10]	QIN T, TIAN Z A, REN H Y, et al. Application of EMA-qPCR as a complementary tool for the detection and monitoring of Legionella in different water systems[J]. World J Microbiol Biotechnol, 2012, 28(5):1881-1890.
[11]	LOPEZ I, RUIZ-LARREA F, COCOLIN L, et al. Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis[J]. Appl Environ Microbiol, 2003, 69(11):6801-6807.
[12]	刘志云, 钟晓霞,谢跃伟,等. 发酵玉米-豆粕型全价饲料对生长猪生长性能、粪便臭味物质和菌群区系的影响[J]. 畜牧兽医学报, 2018, 49(6):1169-1177.
	LIU Z Y, ZHONG X X, XIE Y W, et al. Effects of fermented corn-soybean complete feed on the growth performance, odor concentration and bacterial community in faeces of growing pigs[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(6):1169-1177. (in Chinese)
[13]	李永凯, 毛胜勇,朱伟云. 益生菌发酵饲料研究及应用现状[J]. 畜牧与兽医, 2009, 41(3):90-93.
	LI Y K, MAO S Y, ZHU W Y. Current situation in research and application of fermented feed with probiotics[J]. Animal Husbandry & Veterinary Medicine, 2009, 41(3):90-93. (in Chinese)
[14]	马艳琳, 徐振波,刘君彦,等. EMA-PCR法快速检测啤酒中腐败短乳杆菌[J]. 食品科学, 2017, 38(8):271-276.
	MA Y L, XU Z B, LIU J Y, et al. Development of an ethidium bromide monoazide-polymerase chain reaction assay for raid detection of the beer Spoilage Bacterium Lactobacillus brevis[J]. Food Science, 2017, 38(8):271-276. (in Chinese)
[15]	高延玲, 狄元冉,李金磊,等. EMA与PCR结合检测大肠杆菌O157方法的研究[J]. 畜牧与饲料科学, 2014, 35(10):9-12.
	GAO Y L, DI Y R, LI J L, et al. Study on detection method of E.coli O157 by EMA combined with PCR[J]. Animal Husbandry and Feed Science, 2014, 35(10):9-12. (in Chinese)
[16]	颜成英, 汤晓艳,康大成,等. EMA RT-PCR法检测鸡肉中鼠伤寒沙门氏菌活菌[J]. 中国食品学报, 2015, 15(10):179-184.
	YAN C Y, TANG X Y, KANG D C, et al. Detection of viable salmonella typhimurium in chicken by EMA RT-PCR[J]. Journal of Chinese Institute of Food Science and Technology, 2015, 15(10):179-184. (in Chinese)
[17]	周大祥, 殷幼平,王中康,等. 利用EMA-qPCR建立快速检测猕猴桃溃疡病菌活菌的方法[J]. 植物保护, 2017, 43(3):143-148.
	ZHOU D X, YIN Y P, WANG Z K, et al. Establishment of a method to rapidly detect only viable cells of Pseudomonas syringae pv. actinidiae by EMA-qPCR[J]. Plant Protection, 2017, 43(3):143-148. (in Chinese)
[18]	张俊彦, 梅玲玲,徐昌平,等. EMA实时荧光PCR技术检测食品中单核增生李斯特活菌方法研究[J]. 中国人兽共患病学报, 2017, 33(11):1007-1012.
	ZHANG J Y, MEI L L, XU C P, et al. New EMA real-time fluorescence PCR method for detection of alive Listeria monocytogenes in foods[J]. Chinese Journal of Zoonoses, 2017, 33(11):1007-1012. (in Chinese)

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

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