一种用于芽孢杆菌代谢物细胞毒性的快速检测方法

doi:10.13304/j.nykjdb.2021.0087

摘要/Abstract

摘要：

为建立一种快速检测芽孢杆菌细胞毒性的方法，通过比较不同的细胞以及活性测定方法，确立以非洲绿猴肾细胞（Vero细胞）为受试细胞，MTT比色法作为芽孢杆菌代谢物细胞毒性的检测方法，并对检测条件进行了优化。利用优化后的方法对81株不同来源芽孢杆菌的细胞毒性进行检测，发现有16株芽孢杆菌具有细胞毒性。通过毒素编码基因PCR、溶血性试验和抗体免疫反应试验进一步验证了16株芽胞杆菌的毒性。与体外测定方法相比，建立的细胞毒性检测方法具有更高的灵敏性，且适用于不同株系芽孢杆菌在细胞水平的安全性评价，具有较高的推广应用价值。

关键词: 芽孢杆菌, 毒素, 细胞毒性, MTT比色法

Abstract:

To establish a method for the detection of Bacillus cytotoxicity， the different cells and detection methods were compared. Firstly， African green monkey kidney cell （Vero cells） was as the test cell， MTT colorimetry was selected for detecting the cytotoxicity of Bacillus spp.， and the conditions of cytotoxicity test were optimum. The cytotoxicity of 81 Bacillus strains from different sources was detected using optimum method. The results showed that 16 strains of 81 Bacillus strains were cytotoxic， which were verified by PCR of virulence coden gene detection， hemolysis test and antibody immune response. Compared with those methods in vitro，the Bacillus cytotoxicity detection method established in this study had higher sensitivity and been suitable for the safety evaluation of different strains of Bacillus species at the cell level， which had a high value for popularization and application.

Key words: Bacillus spp., toxin, cytotoxicity, MTT colorimetry

中图分类号:

黄英, 彭晴, 王荃, 徐小轻, 张宇微, 马蓝, 石波, 乔宇. 一种用于芽孢杆菌代谢物细胞毒性的快速检测方法[J]. 中国农业科技导报, 2022, 24(1): 202-210.

Yin HUANG, Qing PENG, Quan WANG, Xiaoqing XU, Yuwei ZHANG, Lan MA, Bo SHI, Yu QIAO. [J]. Journal of Agricultural Science and Technology, 2022, 24(1): 202-210.

图/表 6

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基因名称 Gene name	引物序列 Primer sequence （5’-3’）	片段长度 Fragment length/bp	退火温度 Annealing temperature/℃
ces	F： GGTGACACATTATCATATAAGGTG	1 271	54
ces	R： GTAAGCGAACCTGTCTGTAACAACA	1 271	54
entFM	F： ATGAAAAAAGTAATTTGCAGG	1 269	55
entFM	R： TTAGTATGCTTTTGTGTAACC	1 269	55
CytK	F： CGACGTCACAAGTTGTAACA	565	54
CytK	R： CGTGTGTAAATACCCAGTT	565	54
hblA	F： AAGCAATGGAATACAATGGG	1 154	56
hblA	R： AGAATCTAAATCATGCCACTGC	1 154	56
hblC	F： GATACTAATGTGGCAACTGC	740	58
hblC	R： TTGAGACTGCTCGTTAGTTG	740	58
hblD	F： ACCGGTAACACTATTCATGC	829	58
hblD	R： GAGTCCATATGCTTAGATGC	829	58
nheA	F： GTTAGGATCACAATCACCGC	755	56
nheA	R： ACGAATGTAATTTGAGTCGC	755	56
nheB	F： TTTAGTAGTGGATCTGTACGC	743	54
nheB	R： TTAATGTTCGTTAATCCTGC	743	54
nheC	F： CGGTAGTGATTGCTGGG	564	55
nheC	R： CAGCATTCGTACTTGCCAA	564	55

基因名称 Gene name	引物序列 Primer sequence （5’-3’）	片段长度 Fragment length/bp	退火温度 Annealing temperature/℃
ces	F： GGTGACACATTATCATATAAGGTG	1 271	54
ces	R： GTAAGCGAACCTGTCTGTAACAACA	1 271	54
entFM	F： ATGAAAAAAGTAATTTGCAGG	1 269	55
entFM	R： TTAGTATGCTTTTGTGTAACC	1 269	55
CytK	F： CGACGTCACAAGTTGTAACA	565	54
CytK	R： CGTGTGTAAATACCCAGTT	565	54
hblA	F： AAGCAATGGAATACAATGGG	1 154	56
hblA	R： AGAATCTAAATCATGCCACTGC	1 154	56
hblC	F： GATACTAATGTGGCAACTGC	740	58
hblC	R： TTGAGACTGCTCGTTAGTTG	740	58
hblD	F： ACCGGTAACACTATTCATGC	829	58
hblD	R： GAGTCCATATGCTTAGATGC	829	58
nheA	F： GTTAGGATCACAATCACCGC	755	56
nheA	R： ACGAATGTAATTTGAGTCGC	755	56
nheB	F： TTTAGTAGTGGATCTGTACGC	743	54
nheB	R： TTAATGTTCGTTAATCCTGC	743	54
nheC	F： CGGTAGTGATTGCTGGG	564	55
nheC	R： CAGCATTCGTACTTGCCAA	564	55

菌株名称Strain	粗毒素体积 Volume of crude toxin /μL
菌株名称Strain	25	50	75	100
蜡样芽孢杆菌CICC 21261 B. cereus CICC 21261	93.41±0.16 b	93.69±0.07 a	93.41±0.13 ab	93.54±0.09 ab
蜡样芽胞杆菌1-20 B. cereus 1-20	91.47±0.27 c	94.26±0.04 a	92.84±0.44 b	93.45±0.22 b
解淀粉芽孢杆菌1-16 B. amyloliquefaciens 1-16	50.00±0.39 b	52.64±1.62 a	50.94±0.25 ab	50.96±0.36 ab

菌株名称Strain	粗毒素体积 Volume of crude toxin /μL
菌株名称Strain	25	50	75	100
蜡样芽孢杆菌CICC 21261 B. cereus CICC 21261	93.41±0.16 b	93.69±0.07 a	93.41±0.13 ab	93.54±0.09 ab
蜡样芽胞杆菌1-20 B. cereus 1-20	91.47±0.27 c	94.26±0.04 a	92.84±0.44 b	93.45±0.22 b
解淀粉芽孢杆菌1-16 B. amyloliquefaciens 1-16	50.00±0.39 b	52.64±1.62 a	50.94±0.25 ab	50.96±0.36 ab

编号 Number	菌株 Strain	细胞抑制率 Inhibition rates of cells/%		溶血性 Hemolysis	抗体检测 Antibody test	毒素编码基因 Toxin coding gene
编号 Number	菌株 Strain	正常处理 Normal	热处理 Heat	溶血性 Hemolysis	抗体检测 Antibody test	Ces	entFM	CytK	hblA	hblC	hblD	nheA	nheB	nheC
1	蜡样芽孢杆菌1-13 B. cereus 1-13	93.57±0.11 a	26.32±1.77 b	+	+	-	+	+	+	+	+	+	+	+
2	蜡样芽胞杆菌1-19 B. cereus 1-19	93.60±0.05 a	25.48±0.81 b	+	+	-	+	-	+	+	+	+	+	+
3	蜡样芽胞杆菌1-20 B. cereus 1-20	94.26±0.04 a	3.95±0.63 b	+	+	-	+	+	+	+	+	-	+	+
4	枯草芽孢杆菌2-13 B．subtilis 2-13	54.00±0.42 a	48.63±0.63 b	-	-	-	-	-	-	-	+	-	-	-
5	枯草芽孢杆菌2-14 B．subtilis 2-14	54.30±0.82 a	53.83±0.52 a	-	-	-	-	-	-	-	+	-	-	-
6	枯草芽孢杆菌2-17 B．subtilis 2-17	65.38±1.40 a	50.00±0.31 b	-	-	-	-	-	-	-	+	-	-	-
7	解淀粉芽孢杆2-15 B. amyloliquefaciens 2-15	43.67±0.73 a	45.93±0.59 b	-	-	-	-	-	-	-	+	-	-	-
8	解淀粉芽孢杆菌3-29 B. amyloliquefaciens 3-29	33.29±0.90 a	28.19±1.82 b	-	-	-	-	-	-	-	+	-	-	-
9	解淀粉芽孢杆菌1-16 B. amyloliquefaciens 1-16	52.15±0.58 a	50.78±0.29 a	-	-	-	-	-	-	-	+	-	-	-
10	解淀粉芽孢杆菌1-17 B. amyloliquefaciens 1-17	52.07±0.50 a	51.82±0.68 b	-	-	-	-	-	-	-	-	-	-	-
11	解淀粉芽孢杆菌1-18 B. amyloliquefaciens 1-18	55.32±0.58 a	54.82±0.80 a	-	-	-	-	-	-	-	+	-	-	-
12	解淀粉芽孢杆菌3-33 B. amyloliquefaciens 3-33	62.91±0.90 a	50.09±2.50 b	-	-	-	-	-	-	-	+	-	-	-
13	贝莱斯芽孢杆菌2-16 B．velezensis 2-16	51.60±0.07 a	50.64±1.30 a	-	-	-	-	-	-	-	-	-	-	-
14	贝莱斯芽孢杆菌3-31 B．velezensis 3-31	63.29±0.44 a	57.23±0.95 b	-	-	-	-	-	-	-	+	-	-	-
15	贝莱斯芽孢杆菌3-32 B．velezensis 3-32	55.30±1.48 a	38.41±1.91 b	-	-	-	-	-	-	-	-	-	-	-
16	贝莱斯芽孢杆菌3-34 B．velezensis 3-34	55.26±0.67 a	41.33±0.88 b	-	-	-	-	-	-	-	-	-	-	-