SD-PMA-qPCR快速检测冷冻肉制品中活性沙门氏菌的研究

Rapid Detection of Viable Salmonella spp. in Frozen Meat Products Using Sodium Deoxycholate, Propidium Monoazide, and Real-time Quantitative PCR

摘要:

为实现冷冻肉制品中活性沙门氏菌的快速检测与控制，本文利用PMA（叠氮溴化丙锭）和SD（脱氧胆酸钠）消除死菌和损伤菌的影响，建立了运用SD-PMA-qPCR法检测活性沙门氏菌的反应体系和反应条件，并进行人工染菌样品检测试验。SD的最佳浓度确定为0.1%，最佳孵育时间为20 min。对-20 ℃低温冷冻处理3~4 d的沙门氏菌处理液，使用平板计数法、qPCR、PMA-qPCR和SD-PMA-qPCR进行计数，结果发现qPCR与PMA-qPCR二者检测值接近且明显高于平板计数值，而SD-PMA-qPCR检测值与平板计数值相近，这表明SD和PMA的联合使用能有效的消除死菌和损伤菌的影响。人工染菌样品试验表明，沙门氏菌检出限在102 CFU/g，同时106 CFU/g大肠杆菌O157:H7的存在不会影响检测结果。本研究所建立的SD-PMA-qPCR检测方法特异性好、灵敏度高，有望成为快速检测冷冻肉制品中活性沙门氏菌的新方法，具有很好的研究价值和应用前景。

Abstract:

In order to realize rapid detection of viable Salmonella spp. in frozen meat products, a novel sodium deoxycholate-propidium monoazide-quantitative polymerase chain reaction (SD-PMA-qPCR) method was established using PMA and SD to eliminate interference from dead and injured cells. The reaction conditions were optimized, and an artificially infected sample was tested. The results showed that the optimal SD concentration was 0.1% and the optimal incubation time was 20 min. The numbers of bacterial survivors were compared using plate count, qPCR, PMA-qPCR, and SD-PMA-qPCR assays after the cell suspensions were cryogenically frozen at -20℃ for 3~4 d. The results showed that the numbers of viable Salmonella spp. obtained from qPCR and PMA-qPCR were similar and significantly higher than that from the plate count method. The result from SD-PMA-qPCR was close to that from the plate count method, indicating that a combination of PMA and SD could effectively eliminate the impact of dead and injured bacteria. The artificial infection test results showed that the detection limit of Salmonella spp. was 102 CFU/g, and the presence of 106 CFU/g Escherichia coli O157:H7 did not affect the measurement results. The SD-PMA-qPCR method developed in this study has good specificity and high sensitivity. It is expected to be used as a new method for the rapid detection of viable Salmonella spp. in frozen meat products, and has good research value and application prospects.