沙门菌PCR快速检测试剂盒的研制与应用

Development and application of rapid PCR detection kit for Salmonella

为建立一种简单、快速、灵敏、准确的沙门菌检测方法,根据沙门菌fimY保守基因序列设计合成了1对引物,建立了快速检测沙门菌的PCR方法,并对反应条件进行优化,组装成快速检测试剂盒。结果显示,所有沙门菌均扩增出526 bp的特异性条带,而非沙门菌均未扩增出任何条带。检测灵敏度达93 CFU,还可检出含3 CFU的样品用BP预增菌的菌液或用MM增菌后的菌液,而且稳定性良好,冷冻至少能保存12个月。采用直接菌体加入法、热裂解法、反复冻融法、CTAB碱裂解法和柱式试剂盒提取法分别提取核酸模板,结果CTAB法效果最好,但操作烦琐,而直接菌体加入法和热裂解法可以达到和柱式试剂盒提取法相同的效果,且操作简便、快速、经济、效果好。通过对临床样品的检测,证实该试剂盒具有操作简便、快速、灵敏度高、特异性强、重复性好、稳定性高等优点,值得推广应用。

To establish a simple, sensitive,accurate and rapid PCR method for detection of Salmonella in foods, one pair of specific primers were designed and synthesized according to the conserved gene fimY of Salmonella available in GenBank, and reaction parameters were optimized to develop a PCR detection kit. The fimY gene of 526 bp was amplified in all detected Salmonella by the PCR and no any fragments in 11 non-Salmonella strains. The sensitivity of PCR was 93 CFU and it could be used to detect pre-cultured （BP） or cultured（MM） bacteria liquid of the samples containing 3 CFU Salmonella. It could be stored over 12 months at --20 ℃. The nucleic acid was purified by five methods,including adding bacteria, hot cleavage,freezing-thaw repeatedly,CTAB and column reagent kit. The CTAB method is the best,but its procedure is multifarious. The adding bacteria and the hot cleavage methods can reach the same result as the column reagent kit and are simple,rapid and economical, and are worthy be to applied. The results showed that the developed kit is rapid, simple, sensitive, specific, accurate and easy for detection of clinical samples with Salmonella and can be applied for the detection of a large number of samples.