食品中沙门氏菌分子检测靶点的筛选与评价

Evaluation of new specific molecular targets for the PCR detection of Salmonella spp. in foods

摘要:

【目的】发掘新的沙门氏菌分子检测靶点，筛选检测性能优秀的引物。【方法】利用BLAST程序比较沙门氏菌属内基因组DNA序列的同源性以及沙门氏菌与非沙门氏菌基因组DNA序列之间的特异性，发掘出100多个检测沙门氏菌属的特异性片段，并从中随机挑选出15个片段作为候选靶点，一共设计了27对引物（FS1~FS27），对它们的特异性、灵敏度加以评价，从中筛选检测性能最好的引物。【结果】在27对引物中，检测性能最优的引物为FS23，采用该引物对供试菌株的相应检测靶点进行PCR扩增，44株沙门氏菌都能扩增到一条492 bp特异性片段，而22株非沙门氏菌则不能扩增出这一特异性片段。以FS23为引物建立PCR方法检测猪霍乱沙门氏菌基因组DNA 的灵敏度为11.9 fg/mL，细菌纯培养物灵敏度为4.9×102 cfu/mL；用猪霍乱沙门氏菌人工污染牛奶样品，如果接种起始菌量为100 cfu/25 mL时，只需要增菌5 h，采用上述方法即能检测出沙门氏菌。【结论】引物FS23对应的基因序列是一个性能优良的新分子检测靶点，具备很高的特异性和灵敏性，能够广泛应用于食品中沙门氏菌的快速检测。

Abstract:

[Objective] The purpose of this study is to find new molecular targets for the detection of Salmonella. [Methods] Homology comparison of genomic sequences in GenBank among Salmonella serovars and the specificity comparison among Salmonella and non-Salmonella strains were done with the BLAST (Basic Local Alignment Search Tool Program). We randomly selected 15 target sequences to design and synthesize 27 sets of primers for the evaluation of specificity and sensitivity and development of PCR methods. [Results] Primer FS23 was the best in specificity and sensitivity among these 27 sets of primers. The 44 Salmonella strains produced a specific 492-bp amplification band, whereas 22 non- Salmonella strains did not using primer FS23 for PCR detection. The detection sensitivity of FS23 was 11.9 fg/mL for DNA templates and 4.9×102 cfu/mL for whole cells. Salmonella could be detected successfully by the PCR method developed in this study after 5 h enrichment when the milk samples were artificially contaminated by this organism at 100 cfu/25 mL. [Conclusion] The sequences against FS23 is a new and excellent molecular target for the detection of Salmonella. The FS23-based PCR assay is sensitive and specific, and can be used for rapid detection of Salmonella in foods.