沙门氏菌特异性靶点的筛选及荧光定量PCR检测方法的建立
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摘要
本研究利用生物信息学手段,通过比对沙门氏菌及其他原核生物的基因组序列,发掘出361段沙门氏菌特异性片段作为候选分子检测靶点。从这些序列中随机挑选出30个序列片段,设计普通PCR引物,并用58株沙门氏菌菌株和22株非沙门氏菌菌株对每对引物的检测特异性进行评价,筛选出15对特异性较好的引物。经进一步灵敏度评价,得到6对检测灵敏度较好的引物,分别为:c1(36.5 fg/PCR,500 cfu/ mL);c3(36.5 fg/PCR,500 cfu/ mL);c20(36.5 fg/PCR,500 cfu/ mL);c23(36.5 fg/PCR,5×104 cfu/ mL);c24(36.5 fg/PCR,500 cfu/ mL)和plc(36.5 fg/PCR,500 cfu/ mL)。用鼠伤寒沙门氏菌(ATCC 14028)人为污染牛奶样品后,用建立好的普通PCR方法进行检测验证,当初始接种菌量为8 cfu/25g牛奶时,引物c3,c20,c24和plc均能在增菌6-8小时内检出阳性结果;而严重污染的牛奶样品(>104 cfu/25 g牛奶)则只需增菌2-4小时即可检出沙门氏菌。
在普通PCR方法的基础上,挑选出一段特异性较好的沙门氏菌基因组片段,设计出MGB-TaqMan探针p4以及相应的引物。同时,利用DNA随机重组技术构建扩增内标探针,合成单链寡核苷酸直接作为内标模板,以指示由PCR抑制剂等因素引起的假阴性结果,建立沙门氏菌实时荧光PCR检测方法,并对其特异性、灵敏度、抗干扰性以及在食品样品中的实际检测能力进行评价。经40株沙门氏菌和24株非沙门氏菌检测验证,荧光定量PCR具有良好的特异性,以肠炎沙门氏菌(CDC H3526)基因组DNA作为模板,检测灵敏度达到18.6 fg/PCR;以鼠伤寒沙门氏菌(CDC G7601)基因组DNA作模版,PCR反应检测灵敏度为40.2 fg/PCR。在鸡肉样品中自然存在背景菌群的干扰下,PCR检测限为130 cfu/mL,且PCR定量结果与实际接入的沙门氏菌量处于相同或相近水平。用所建立的荧光定量PCR方法检测人工污染至鸡肉样品中的沙门氏菌,当污染量低至5 cfu/10 g样品时,经6小时非选择性增菌后,检验结果与标准方法符合率为23/24(95.8%);人工污染液体鸡蛋样品,初始接种量低至1 cfu/10 g样品时,检出率为100%,并与标准检测方法结果吻合;荧光定量PCR方法对人工污染沙门氏菌的花生酱样品检出率为26/27(96.3%),与标准方法符合率为35/36(97.2%),检测限低至3 cfu/ 10g样品。
In this study 361 candidates for Salmonella-specific targets were successfully identified by comparing the sequences of Salmonella enterica and other prokaryotic genomes using bio-informatic analysis. Primers were designed from 30 randomly selected fragments for end-point PCR detection. The specificity of 15 primer sets was verified by testing against 58 Salmonella strains and 22 non-Salmonella strains, and 6 primer pairs, namely c1 (36.5 fg/PCR, 500 cfu/ mL); c3 (36.5 fg/PCR, 500 cfu/ mL); c20 (36.5 fg/PCR, 500 cfu/ mL); c23 (36.5 fg/PCR, 5×104 cfu/ mL); c24 (36.5 fg/PCR,500 cfu/ mL) and plc (36.5 fg/PCR, 500 cfu/ mL),showed high sensitivity. The PCR method yielded positive results from milk samples that were artificially contaminated with Salmonella Typhimurium (as low as 8 cfu/25mL milk) within 6-8 hours of non-selective enrichment. The enrichment time was shortened to 2-4 hours when the samples were heavily contaminated ( >104 cfu/25mL milk).
One of the verified Salmonella specific fragments was targeted to generate a minor groove binding TaqMan probe and the corresponding primer set for real-time PCR detection. An internal amplification control obtained by DNA random shuffling technology was included in the real-time PCR assay to indicate false negative results caused by PCR inhibitors. The real-time PCR method, incorporated with the IAC, demonstrated a high specificity when tested against 40 Salmonella strains and 24 non-Salmonella strains. Detection limits were 18.6 fg genomic DNA /PCR for Salmonella Enteritidis (CDC H3526) and 40.2 fg/PCR for Salmonella Typhimurium (CDC G7601). In the presense of a natural background flora enriched from chicken breast samples, the detection limit was as low as 130 cfu/mL, and the quantitation results of the real-time PCR assay were in accordance with the actual amount of the spiked Salmonella cells prior to DNA extraction. The real-time PCR method was able to detect as low as 5 cfu/10g chicken sample or 1 cfu/ 10g liquid egg sample after a 6-hour non-selective enrichment. Twenty-six out of 27 peanut butter samples, artificially contaminated with as low as 3 cfu/10g sample, were confirmed positive by this real-time PCR sysytem, exhibiting a 97.2% consistency with the standard culture methods.
在普通PCR方法的基础上,挑选出一段特异性较好的沙门氏菌基因组片段,设计出MGB-TaqMan探针p4以及相应的引物。同时,利用DNA随机重组技术构建扩增内标探针,合成单链寡核苷酸直接作为内标模板,以指示由PCR抑制剂等因素引起的假阴性结果,建立沙门氏菌实时荧光PCR检测方法,并对其特异性、灵敏度、抗干扰性以及在食品样品中的实际检测能力进行评价。经40株沙门氏菌和24株非沙门氏菌检测验证,荧光定量PCR具有良好的特异性,以肠炎沙门氏菌(CDC H3526)基因组DNA作为模板,检测灵敏度达到18.6 fg/PCR;以鼠伤寒沙门氏菌(CDC G7601)基因组DNA作模版,PCR反应检测灵敏度为40.2 fg/PCR。在鸡肉样品中自然存在背景菌群的干扰下,PCR检测限为130 cfu/mL,且PCR定量结果与实际接入的沙门氏菌量处于相同或相近水平。用所建立的荧光定量PCR方法检测人工污染至鸡肉样品中的沙门氏菌,当污染量低至5 cfu/10 g样品时,经6小时非选择性增菌后,检验结果与标准方法符合率为23/24(95.8%);人工污染液体鸡蛋样品,初始接种量低至1 cfu/10 g样品时,检出率为100%,并与标准检测方法结果吻合;荧光定量PCR方法对人工污染沙门氏菌的花生酱样品检出率为26/27(96.3%),与标准方法符合率为35/36(97.2%),检测限低至3 cfu/ 10g样品。
In this study 361 candidates for Salmonella-specific targets were successfully identified by comparing the sequences of Salmonella enterica and other prokaryotic genomes using bio-informatic analysis. Primers were designed from 30 randomly selected fragments for end-point PCR detection. The specificity of 15 primer sets was verified by testing against 58 Salmonella strains and 22 non-Salmonella strains, and 6 primer pairs, namely c1 (36.5 fg/PCR, 500 cfu/ mL); c3 (36.5 fg/PCR, 500 cfu/ mL); c20 (36.5 fg/PCR, 500 cfu/ mL); c23 (36.5 fg/PCR, 5×104 cfu/ mL); c24 (36.5 fg/PCR,500 cfu/ mL) and plc (36.5 fg/PCR, 500 cfu/ mL),showed high sensitivity. The PCR method yielded positive results from milk samples that were artificially contaminated with Salmonella Typhimurium (as low as 8 cfu/25mL milk) within 6-8 hours of non-selective enrichment. The enrichment time was shortened to 2-4 hours when the samples were heavily contaminated ( >104 cfu/25mL milk).
One of the verified Salmonella specific fragments was targeted to generate a minor groove binding TaqMan probe and the corresponding primer set for real-time PCR detection. An internal amplification control obtained by DNA random shuffling technology was included in the real-time PCR assay to indicate false negative results caused by PCR inhibitors. The real-time PCR method, incorporated with the IAC, demonstrated a high specificity when tested against 40 Salmonella strains and 24 non-Salmonella strains. Detection limits were 18.6 fg genomic DNA /PCR for Salmonella Enteritidis (CDC H3526) and 40.2 fg/PCR for Salmonella Typhimurium (CDC G7601). In the presense of a natural background flora enriched from chicken breast samples, the detection limit was as low as 130 cfu/mL, and the quantitation results of the real-time PCR assay were in accordance with the actual amount of the spiked Salmonella cells prior to DNA extraction. The real-time PCR method was able to detect as low as 5 cfu/10g chicken sample or 1 cfu/ 10g liquid egg sample after a 6-hour non-selective enrichment. Twenty-six out of 27 peanut butter samples, artificially contaminated with as low as 3 cfu/10g sample, were confirmed positive by this real-time PCR sysytem, exhibiting a 97.2% consistency with the standard culture methods.
引文
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[5] Abubakar, I.; Irvine, L.; Aldus, C. F., etc., A systematic review of the clinical, public health and cost-effectiveness of rapid diagnostic tests for the detection and identification of bacterial intestinal pathogens in faeces and food[J]. Health Technology Assessment. 2007, 11 (36), 1-216.
[6]陈建林;刘明辉,细菌性食物中毒流行趋势及预防对策[J].中国卫生检验杂志. 2002, 12 (4), 480-481.
[7] Manzano, M.; Cocolin, L.; Astori, G., etc., Development of a PCR microplate-capture hybridization method for simple, fast and sensitive detection of Salmonella serovars in food[J]. Molecular and Cellular Probes. 1998, 12 (4), 227-234.
[8] Whyte, P.; Mc Gill, K.; Collins, J. D., etc., The prevalence and PCR detection of Salmonella contamination in raw poultry[J]. Veterinary Microbiology. 2002, 89 (1), 53-60.
[9] CDC, Salmonella Annual Summary[J]. 2005.
[10] Multistate outbreak of Salmonella infections associated with peanut butter and peanut butter-containing products--United States, 2008-2009[J]. Morbidity and Mortality Weekly Report. 2009, 58 (4), 85-90.
[11] -2003, G. T.,食品卫生微生物学检验沙门菌检验[J].中国标准出版社. 2003.
[12] AOAC/BAM(Bacteriological Analytical Manual, FDA).伯杰细菌鉴定手册(第八版)[J].科学出版社. 1984.
[13] Isolation And Identification of Salmonella From Meat, Poultry And Egg Products (MLG 4.04). United States Department of Agriculture, Food Safety and Inspection Service 2008.
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