PMA-qPCR定量检测畜禽肉类中沙门菌活菌的研究

doi:10.3969/j.issn.1673-8640.2015.05.021

摘要/Abstract

摘要： 目的将叠氮溴化丙锭(PMA)与实时荧光定量聚合酶链反应(qPCR)相结合定量检测畜禽肉类中活的沙门菌。方法通过优化光反应时间、PMA浓度等PMA作用条件,建立PMA-qPCR方法,构建重组质粒建立标准曲线,考察该方法的灵敏性、特异性,并将该方法用于定量检测未经增菌培养的畜禽肉类样品中的沙门菌。结果在PMA浓度为15 μg/mL、曝光5 min的条件下可完全抑制样品中死菌DNA的扩增。建立的定量标准曲线循环阈值(Ct值)与质粒标准品模板的拷贝数呈良好线性关系(r²=0.997 9),最低可检出10 拷贝/反应体系。所建立的PMA-qPCR方法最低可检出21 拷贝/μL沙门菌。采用PMA-qPCR检测人工染菌鸡肉样品,最低可检出10³ CFU/mL沙门菌。结论用PMA-qPCR方法可实现定量检测畜禽肉类样品中活的沙门菌,从而达到快速检测的目的。

关键词: 沙门菌, 活菌, 叠氮溴化丙啶, 荧光定量聚合酶链反应, 畜禽肉类

Abstract: Objective To enumerate Salmonella in meat of livestock and poultry rapidly and accurately by using propidium monoazide(PMA) combined with real-time fluorescence quantitation polymerase chain reaction(qPCR). Methods The light exposure time and the concentration of PMA were optimized to establish PMA-qPCR. The standard curve was established by standard plasmid. The sensitivity and specificity were investigated. This method was used for the quantitation determination of Salmonella in livestock and poultry meat. Results The amplification of DNA derived from Salmonella dead cells could be inhibited without affecting the viable cells when PMA was at a dose of 15 μg/mL and exposed for 5 min. The cycle threshold values(Ct) and standard plasmid model cell copy number presented the satisfactory linear, and the correlation coefficient r² approached 0.997 9. This method could detect as low as 10 copies/reaction. The minimum detection level was 21 copies/μL by PMA-qPCR. In artificial chicken samples, PMA-qPCR could detect as low as 10³ CFU/mL. Conclusions It was possible to quantify viable Salmonella in meat of livestock and poultry by PMA-qPCR.

Key words: Salmonella, Viable bacterium, Propidium monoazide, Real-time fluorescence quantitation polymerase chain reaction, Livestock and poultry meat

中图分类号:

R378.2

於颖, 王文静, 陆晔. PMA-qPCR定量检测畜禽肉类中沙门菌活菌的研究[J]. 检验医学, 2015, 30(5): 500-506.

YU Ying, WANG Wenjing, LU Ye. Research on a quantitative method to detect viable Salmonella by PMA-qPCR in livestock and poultry meat[J]. Laboratory Medicine, 2015, 30(5): 500-506.

图/表 12

参考文献 14

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类别	菌株	来源
沙门菌	鼠伤寒沙门菌	ATCC 14028
	肠炎沙门菌	ATCC 49214
非沙门菌	阪崎肠杆菌	ATCC 51329
	大肠埃希菌	ATCC 25922
	铜绿假单胞菌	ATCC 27853
	副溶血弧菌	ATCC 17802
	金黄色葡萄球菌	ATCC 6538
	宋内志贺菌	ATCC 11060
	小肠结肠耶尔森菌	Y1
	单增李斯特菌	总68-88
	大肠埃希菌 O157	总75-86
	创伤弧菌	总18-85

类别	菌株	来源
沙门菌	鼠伤寒沙门菌	ATCC 14028
	肠炎沙门菌	ATCC 49214
非沙门菌	阪崎肠杆菌	ATCC 51329
	大肠埃希菌	ATCC 25922
	铜绿假单胞菌	ATCC 27853
	副溶血弧菌	ATCC 17802
	金黄色葡萄球菌	ATCC 6538
	宋内志贺菌	ATCC 11060
	小肠结肠耶尔森菌	Y1
	单增李斯特菌	总68-88
	大肠埃希菌 O157	总75-86
	创伤弧菌	总18-85

用途	引物与探针	序列(5'-3')	产物长度(bp)
PCR	IM832-F	GTGAAATTATCGCCACGTTCGGGCAA	284^[6]
	IM832-R	TCATCGCACCGTCAAAGGAACC
qPCR	invA-F	CGTTCTACATTGACAGAAT	144^[7]
	invA-R	CGAACGTGGCGATAATTTC
	invA-P	FAM-CAACGTTTCCTGCGGTACTGTTAAT- TAMRA
质粒	invA-S	CGGCAATAGCGTCACCTT	577
	invA-AS	AGTGCTCGTTTACGACCTG

用途	引物与探针	序列(5'-3')	产物长度(bp)
PCR	IM832-F	GTGAAATTATCGCCACGTTCGGGCAA	284^[6]
	IM832-R	TCATCGCACCGTCAAAGGAACC
qPCR	invA-F	CGTTCTACATTGACAGAAT	144^[7]
	invA-R	CGAACGTGGCGATAATTTC
	invA-P	FAM-CAACGTTTCCTGCGGTACTGTTAAT- TAMRA
质粒	invA-S	CGGCAATAGCGTCACCTT	577
	invA-AS	AGTGCTCGTTTACGACCTG

菌液	0%	1%	10%	25%	50%	100%
活菌	0	5.00×10⁶	5.0×10⁷	1×10⁸	2.5×10⁸	5×10⁸
死菌	5×10⁸	4.95×10⁸	4.5×10⁸	4×10⁸	2.5×10⁸	0