畜禽养殖粪污中典型致病菌的三重微滴式数字PCR定量检测方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2022-0944

摘要/Abstract

摘要：

为精准、快速检测畜禽养殖粪污中典型致病微生物,减少人畜共患病的传播风险,以金黄色葡萄球菌、大肠埃希氏菌（O157：H7）和肠炎沙门氏菌3种常见致病菌为研究对象,通过筛选其特异性引物与探针、优化反应系统,建立起快速、稳定的多重微滴式数字PCR（droplet digital PCR,ddPCR）反应体系。通过检测不同菌株验证该体系的特异性,并确定畜禽养殖废弃物致病菌的检出限,开发出多重微滴数字PCR快速检测方法。研究结果表明,各对引物探针对目标菌株均能扩增,ddPCR体系内未出现交叉反应,检测肠炎沙门氏菌的绝对定量检测低限为0.68 copies/µL;检测金黄色葡萄球菌的绝对定量检测低限为0.79 copies/µL;检测大肠埃希氏菌的绝对定量检测低限为1.02 copies/µL。研究建立的方法可实现对畜禽养殖粪污中3种典型致病菌的高效率、高精度的检测。

关键词: 养殖粪污, 沙门氏菌, 大肠埃希氏菌, 金黄色葡萄球菌, 三重微滴式数字PCR

Abstract:

In order to accurately and rapidly detect typical pathogenic microorganisms in livestock and poultry manure and reduce the risk of transmission of zoonotic diseases,three common pathogenic bacteria,Staphylococcus aureus,Escherichia coli（O157：H7）and Salmonella enteritidis,were used as the research object. By screening its specific primers and probes as well as optimizing the reaction system,a fast and stable multiplex droplet digital PCR（ddPCR）reaction system was established. The specificity of the system was verified by detecting different strains,the detection limit of pathogenic bacteria in livestock and poultry waste was determined,and a rapid detection method of multiple droplet digital PCR was developed. The results showed that each pair of primers and probes amplified the target strains without cross-reaction in the ddPCR system. The absolute quantitative lower limit of detecting S. enteritidis was 0.68 copies/µL,and that for S. aureus was 0.79 copies/µL,and that for E. coli was 1.02 copies/µL. The high-efficiency and high-precision detection of three typical pathogenic bacteria in livestock and poultry manure may be achieved by the method established in this study.

Key words: livestock waste, Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, triple droplet digital PCR

程深伟, 张克强, 梁军锋, 刘福元, 郜兴亮, 杜连柱. 畜禽养殖粪污中典型致病菌的三重微滴式数字PCR定量检测方法的建立[J]. 生物技术通报, 2022, 38(9): 271-280.

CHENG Shen-wei, ZHANG Ke-qiang, LIANG Jun-feng, LIU Fu-yuan, GAO Xing-liang, DU Lian-zhu. Establishment of a Triple Droplet Digital PCR Quantitative Detection Method for Typical Pathogenic Bacteria in Livestock and Poultry Manure[J]. Biotechnology Bulletin, 2022, 38(9): 271-280.

图/表 15

表1 3种典型致病菌具体信息

Table 1 Specific information on three typical pathogenic bacteria

菌株名称Species	编号Code	来源Origin
金黄色葡萄球菌	CMCC（B）26073	中国医学细菌保藏管理中心
大肠埃希氏菌（O157：H7）	ATCC 25922	美国菌种保藏中心
肠炎沙门氏菌	CMCC（B）50041	中国医学细菌保藏管理中心

表2 三重数字PCR检测3种致病菌的特异性引物

Table 2 Specific primers for detecting three pathogens by triple droplet digital PCR

菌株名称Species	靶基因Target gene	引物探针序列Primer sequence（5'-3'）
金黄色葡萄球菌	nuc	CCTGAAGCAAGTGCATTTACGA
		CTTTAGCCAAGCCTTGACGAACT
		HEX-TGGACGTGGCTTAGCGTATATTTATGCTGATG-BHQ1
大肠埃希氏菌	rfbE	TCAAAAGGAAACTATATTCAGAAGTTTGA
		CGATATACCTAACGCTAACAAAGCTAA
		CY5-AATAAATTTGCGGAACAAAACCATGTGCAA-BHQ2
肠炎沙门氏菌	invA	GCGGCGTTGGAGAGTGATA
		AGCAATGGAAAAAGCAGGATG
		FAM-CATTTCTTAAACGGCGGTGTCTTTCCCT-BHQ1

图1 肠炎沙门氏菌单重ddPCR检测一维图 1：肠炎沙门氏菌（CMCC（B）50041）;2：空白对照;灰色：阴性微滴;蓝色：阳性微滴

Fig. 1 One-dimensional diagram of single ddPCR detection of S. enteritidis 1：S. enteritidis（CMCC（B）50041）;2：blank control;gray：negative droplets;blue：positive droplets

图2 金黄色葡萄球菌单重ddPCR检测一维图 1：金黄色葡萄球菌（CMCC（B）26073）;2：空白对照;灰色：阴性微滴;绿色：阳性微滴

Fig. 2 One-dimensional diagram of single ddPCR detection of S. aureus 1：S. aureus（CMCC（B）26073）;2：blank control;gray：negative droplets;green：positive droplets

图3 大肠埃希氏菌单重ddPCR检测一维图 1：大肠埃希氏菌（O157：H7）（ATCC 25922）;2：空白对照;灰色：阴性微滴;红色：阳性微滴

Fig. 3 One-dimensional diagram of single ddPCR detection of E.coli 1：E.coli（O157：H7）（ATCC 25922）;2：blank control;gray：negative droplets;red：positive droplets

表3 单重及多重微滴数字PCR检测样本拷贝数结果

Table 3 Copy number of samples detected by single and multiple droplet digital PCR

样品名称 Name of samples	总微滴数 Total number of droplets	肠炎沙门氏菌 S. enteritidis/ （Cp·µL^-1）	阳性微滴 Number of positive droplets	金黄色葡萄球菌 S. aureus/ （Cp·µL^-1）	阳性微滴 Number of positive droplets	大肠埃希氏菌 E. coli/（Cp·µL^-1）	阳性微滴 Number of positive droplets
沙门-阳性	27018	59.9	932	0	0	0	0
金黄-阳性	26349	0	0	75.7	1 143	0	0
大肠-阳性	26983	0	0	0	0	76.8	1 187
三重-阳性	25796	61.6	915	79.2	1 169	81.7	1 206

图4 三重ddPCR特异性检测一维图 A：FAM通道,肠炎沙门氏菌检测一维图;B：HEX通道,金黄色葡萄球菌检测一维图;C：CY5通道,大肠埃希氏菌检测一维图。1：阳性样本;2-11：非目标菌株样本;12：空白对照

Fig. 4 One-dimensional map of specificity detected by triple ddPCR A：FAM channel,one-dimensional map of S. enteritidis detection. B：HEX channel,one-dimensional map of S.aureus detection. C：CY5 channel,one-dimensional map of E.coli detection. 1：Positive sample;2-11：non-target strain sample;12：blank control

表4 三重微滴数字PCR灵敏度检测结果

Table 4 Sensitivity detection results by triple droplet digital PCR

样本稀释 Sample dilution	肠炎沙门氏菌S. enteritidis /（Cp·µL^-1）						金黄色葡萄球菌S. aureus/（Cp·µL^-1）						大肠埃希氏菌E.coli/（Cp·µL^-1）
样本稀释 Sample dilution	Repeat1	Repeat2	Repeat3	AVG	SD	RSD/%	Repeat1	Repeat2	Repeat3	AVG	SD	RSD/%	Repeat1	Repeat2	Repeat3	AVG	SD	RSD/%
DNA-10×	8 324	7 814	8 326	8 154.67	240.89	2.95	8 306	7 572	8 255	8 044.33	334.64	4.16	8 390	7 502	8 460	8 117.33	436.04	5.37
DNA-100×	785.3	702.8	756.9	748.33	34.22	4.57	830.8	774.9	788.1	797.93	23.86	2.99	831.2	755.3	803.1	796.53	31.33	3.93
DNA-1 000×	73.7	65.9	83.8	74.47	7.33	9.84	87.3	85.8	69.8	80.97	7.92	9.78	84	73	76.6	77.87	4.58	5.88
DNA-10 000×	7.67	7.77	5.48	6.97	1.06	15.15	6.21	8.11	8.38	7.57	0.97	12.76	8.41	8.78	5.8	7.66	1.33	17.31
DNA-100 000×	0.35	1.04	0.66	0.68	0.28	41.29	0.35	1.04	0.99	1.02	0.31	39.60	1.06	0.69	1.32	1.02	0.26	25.26
DNA-1 000 000×	0	0	0	0	0	/	0	0	0	0	0	/	0	1.75	0	0.58	0.82	141.42

图5 三重ddPCR灵敏度检测一维图 A：FAM通道,肠炎沙门氏菌检测一维图;B：HEX通道,金黄色葡萄球菌检测一维图;C：CY5通道,大肠埃希氏菌检测一维图

Fig. 5 One-dimensional map of sensitivity detected by triple ddPCR A：The one-dimensional diagram of FAM channel and S. enteritidis detection. B：The one-dimensional diagram of HEX channel and S. aureus detection. C：The one-dimensional diagram of CY5 channel and E.coli detection

图6 三重ddPCR灵敏度检测线性范围拟合曲线 A：肠炎沙门氏菌灵敏度检测线性范围拟合曲线;B：金黄色葡萄球菌灵敏度检测线性范围拟合曲线;C：大肠埃希氏菌灵敏度检测线性范围拟合曲线

Fig. 6 Fitting curve of linear range for triple ddPCR sensitivity detection A：The fitting curve of the linear range of the sensitivity detection of S.enteritidis. B：The fitting curve of the linear range of the sensitivity detection of S.aureus. C：The fitting curve of the linear range of the sensitivity detection of E. coli

表5 16份样本DNA浓度检测结果

Table 5 DNA concentration test results of 16 samples

样品编号 Number of samples	样品名称 Name of samples	A₂₆₀/A₂₈₀	A₂₆₀/A₂₃₀	浓度 Concentration/ （ng·µL^-1）
1	猪1	2.23	0.21	536.8
2	猪2	2.09	0.41	1 127
3	猪3	2.1	0.35	956.4
4	猪4	2.1	0.65	2 110.5
5	猪5	2.21	0.19	455.1
6	猪6	2.11	0.48	1 337.8
7	猪7	2.48	0.12	281.6
8	猪8	2.36	0.13	280.4
9	猪9	1.99	14.66	382.85
10	鸡1	2.21	0.37	972.8
11	鸡2	2.57	0.1	225.4
12	鸡3	2.17	0.75	2 523.2
13	鸡4	2.05	8.49	393.56
14	鸡5	2.72	0.1	240.7
15	鸡6	1.96	10.14	684.53
16	鸡7	2.13	7.95	407.08

表6 16份样本DNA浓度琼脂糖凝胶电泳检测表

Table 6 Detection of DNA concentration in 16 samples by agarose gel electrophoresis

样品编号 Number of samples	样品名称 Name of samples	上样量 Sample amount
M	Marker	3μL
1	猪1	1μL
2	猪2	1μL
3	猪3	1μL
4	猪4	1μL
5	猪5	1μL
6	猪6	1μL
7	猪7	1μL
8	猪8	1μL
9	猪9	1μL
10	鸡1	1μL
11	鸡2	1μL
12	鸡3	1μL
13	鸡4	1μL
14	鸡5	1μL
15	鸡6	1μL
16	鸡7	1μL

图7 16份样本琼脂糖凝胶电泳结果

Fig. 7 Agarose gel electrophoresis results of 16 samples

图8 三重ddPCR检测14样本一维图 A：FAM通道,肠炎沙门氏菌检测一维图;B：HEX通道,金黄色葡萄球菌检测一维图;C：CY5通道,大肠埃希氏菌检测一维图

Fig. 8 One-dimensional diagram of detecting 14 samples by triple ddPCR A：The one-dimensional diagram of FAM channel and S. enteritidis detection. B：The one-dimensional diagram of HEX channel and S. aureus detection. C：The one-dimensional diagram of CY5 channel and E.coli detection.

表7 16份样本三重微滴数字PCR拷贝数检测结果

Table 7 Copy number detection results for 16 samples by triple droplet digital PCR

样品名称 Name of samples	总微滴数 Total number of droplets	肠炎沙门氏菌 S. enteritidis/ （Cp·µL^-1）	阳性微滴 Number of positive droplets	金黄色葡萄球菌 S. aureus/ （Cp·µL^-1）	阳性微滴 Number of positive droplets	大肠埃希氏菌 E. coli/ （Cp·µL^-1）	阳性微滴 Number of positive droplets
猪1	12 494	0	0	0	0	0.34	1
猪2	13 744	0	0	0	0	0	0
猪3	15 670	0	0	0	0	0	0
猪4	14 445	0	0	1.18	4	0.59	2
猪5	13 914	0	0	0	0	0.92	3
猪6	13 173	0	0	0	0	0	0
猪7	13 183	0	0	0	0	0.64	2
猪8	13 367	0	0	0	0	0	0
猪9	14 361	0	0	0	0	0	0
鸡1	12 270	0	0	0	0	0	0
鸡2	13 422	0	0	0	0	0	0
鸡3	14 292	0	0	0	0	0	0
鸡4	13 928	0	0	0	0	0.31	1
鸡5	14 225	0	0	0	0	0	0
鸡6	13 949	0.3	1	0	0	2.13	7
鸡7	13 602	0	0	0	0	2.19	7

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