该研究旨在利用多重巢式聚合酶链式反应(polymerase chain reaction, PCR)与毛细管电泳技术建立快速检测大肠埃希氏菌、鼠伤寒沙门氏菌和金黄色葡萄球菌的方法。该文以大肠埃希氏菌β-葡萄糖苷酶基因(uidA基因)、鼠伤寒沙门氏菌毒力基因(hilA基因)和金黄色葡萄球菌耐热核酸酶基因(nuc基因)为靶标,设计3对嵌套式引物进行2轮扩增,利用毛细管电泳分析产物,通过对2轮巢式PCR退火温度的优化,建立3种食源性致病菌的多重巢式PCR-毛细管电泳检测方法。3对嵌套式引物特异性良好,2轮巢式PCR最佳退火温度分别为54 ℃和56 ℃,对大肠埃希氏菌和金黄色葡萄球菌检测灵敏度可达101 copies,对鼠伤寒沙门氏菌检测灵敏度可达103copies。该方法快速、准确和高灵敏,适用于能提取DNA的食品样本中3种食源性致病菌的检测,为食源性致病菌的检测提供新方案。
This study aimed to establish a rapid detection method for Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus using multiplex nested polymerase chain reaction (PCR) combined with capillary electrophoresis.Targeting the uidA gene of E.coli (β-glucuronidase), the hilA gene of S.typhimurium (virulence gene), and the nuc gene of S.aureus (thermostable nuclease gene), three pairs of nested primers were designed for two rounds of amplification.The PCR products were analyzed by capillary electrophoresis, and the annealing temperatures for the two rounds of nested PCR were optimized to establish a multiplex nested PCR-capillary electrophoresis detection method for the three foodborne pathogens.The three pairs of nested primers showed high specificity, with optimal annealing temperatures of 54 ℃ and 56 ℃ for the two rounds.The detection sensitivity reached 101 copies for E.coli and S.aureus, and 103 copies for S.typhimurium.This method is rapid, accurate, and highly sensitive, making it suitable for detecting these three foodborne pathogens in food samples from which DNA can be extracted, thereby providing a new approach for foodborne pathogen detection.
[1] SAUCIER L. Microbial spoilage, quality and safety within the context of meat sustainability[J]. Meat Science, 2016, 120:78-84.
[2] MEAD P S, SLUTSKER L, DIETZ V, et al. Food-related illness and death in the United States[J]. Emerging Infectious Diseases, 1999, 5(5):607-625.
[3] SUN F L, CAO Z S, QIU Z G, et al. Development of a simultaneous identification method for nine species of foodborne bacterial pathogens using real-time PCR assays and melting curve analysis[J]. Journal of Biotech Research, 2020, 11: 73-81.
[4] FODDAI A C G, GRANT I R. Methods for detection of viable foodborne pathogens: Current state-of-art and future prospects[J]. Applied Microbiology and Biotechnology, 2020, 104(10):4281-4288.
[5] ALTAYB H N, BADRI R M, CHAIEB K, et al. Detection and characterization of the most common foodborne pathogens by using multiplex PCR procedure[J]. Saudi Journal of Biological Sciences, 2023, 30(6):103653.
[6] 殷淑权. 多重PCR在食源性微生物快速检验中的效果分析[J]. 智慧健康, 2023, 9(27):207-210.
YIN S Q. Effect analysis of multiplex PCR in rapid test of foodborne microorganisms[J]. Smart Healthcare, 2023, 9(27):207-210.
[7] 李雪艳, 魏彩姣, 王庄舒, 等. 多重PCR检测技术和传统培养法在食源性致病菌检测中的应用分析[J]. 质量与认证, 2025(1):96-98.
LI X Y, WEI C J, WANG Z S, et al. Application analysis of multiplex PCR detection technology and traditional culture method in detection of food-borne pathogens[J]. China Quality Certification, 2025(1):96-98.
[8] LAW J W, AB MUTALIB N S, CHAN K G, et al. Rapid methods for the detection of foodborne bacterial pathogens: Principles, applications, advantages and limitations[J]. Frontiers in Microbiology, 2015, 5:770.
[9] 李聪, 刘健慧, 李志辉, 等. 4种食源性致病菌的多重PCR快速检测方法研究[J]. 食品研究与开发, 2019, 40(21):164-169.
LI C, LIU J H, LI Z H, et al. Study on multiplex PCR of rapid detection method for four foodborne pathogens[J]. Food Research and Development, 2019, 40(21):164-169.
[10] CHAMBERLAIN J S, GIBBS R A, RANIER J E, et al. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification[J]. Nucleic Acids Research, 1988, 16(23):11141-11156.
[11] MARKOULATOS P, SIAFAKAS N, MONCANY M. Multiplex polymerase chain reaction: A practical approach[J]. Journal of Clinical Laboratory Analysis, 2002, 16(1):47-51.
[12] GOULART L R, COLOMBO B F M, LIMA M I S, et al. Expanded HPV genotyping by single-tube nested-multiplex PCR may explain HPV-related disease recurrence[J]. Microorganisms, 2024, 12(11):2326.
[13] 曾成鸣, 康格非. 高效毛细管电泳及其应用进展[J]. 国外医学.临床生物化学与检验学分册, 1995, 16(4):178-179.
ZENG C M, KANG G F. High performance capillary electrophoresis and its application progress[J]. Foreign Medical Sciences, 1995, 16(4):178-179.
[14] 张明娟, 王娟, 袁磊, 等. 多重聚合酶链式反应技术在食源性致病菌检测上的应用研究进展[J]. 食品与发酵工业, 2021, 47(2):305-310.
ZHANG M J, WANG J, YUAN L, et al. Application of multiplex polymerase chain reaction in detection of foodborne pathogens[J]. Food and Fermentation Industries, 2021, 47(2):305-310.
[15] 向婧姝, 周倩, 周藜, 等. 三种多重PCR技术检测食源性致病菌的对比研究[J]. 微量元素与健康研究, 2024, 41(5):59-62.
XIANG J S, ZHOU Q, ZHOU L, et al. Comparative study on detection of food-borne pathogens by three multiplex PCR techniques[J]. Studies of Trace Elements and Health, 2024, 41(5):59-62.
[16] THALINGER B, PÜTZ Y, TRAUGOTT M. Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions[J]. PLoS One, 2021, 16(7): e0254356.
[17] 马佳睿. 食源性金黄色葡萄球菌和沙门氏菌双重巢式PCR及双重LAMP检测方法的建立及应用[D]. 银川: 宁夏大学, 2021.
MA J R. Establishment and application of double nested PCR and double LAMP detection methods to food-borne Staphylococcus aureus and Salmonella[D]. Yinchuan: Ningxia University, 2021.
[18] PODOLAK R, BLACK D G. Control of Salmonella and Other Bacterial Pathogens in Low Moisture Foods[M]. 1st ed. Hoboken, New Jersey: Wiley Blackwell, 2017.
[19] OLIVER J D. Recent findings on the viable but nonculturable state in pathogenic bacteria[J]. FEMS Microbiology Reviews, 2010, 34(4):415-425.
[20] LEI I F, ROFFEY P, BLANCHARD C, et al. Development of a multiplex PCR method for the detection of six common foodborne pathogens[J]. Journal of Food and Drug Analysis, 2008, 16(4): 6.
[21] SHARIFDINI M, MIRHENDI H, ASHRAFI K, et al. Comparison of nested polymerase chain reaction and real-time polymerase chain reaction with parasitological methods for detection of Strongyloides stercoralis in human fecal samples[J]. The American Journal of Tropical Medicine and Hygiene, 2015, 93(6):1285-1291.
[22] 范宏伟, 朱应飞, 翟平平, 等. 多重聚合酶链式反应-毛细管电泳技术检测5种食源性致病菌[J]. 食品安全质量检测学报, 2025, 16(1):180-186.
FAN H W, ZHU Y F, ZHAI P P, et al. Detection of 5 kinds of foodborne pathogens by multiplex polymerase chain reaction and capillary electrophoresis technology[J]. Journal of Food Safety & Quality, 2025, 16(1):180-186.
[23] 朱安基, 李世宏, 徐浩, 等. 奶牛乳房炎大肠埃希氏菌和肺炎克雷伯菌及奇异变形杆菌三重TaqMan荧光定量PCR检测方法的建立[J]. 动物医学进展, 2024, 45(12): 19-25.
ZHU A J, LI S H, XU H, et al. Establishment of a triple TaqMan fluorescence quantitative PCR method for detection of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in dairy mastitis[J]. Progress in Veterinary Medicine, 2024, 45(12): 19-25.
[24] RUANO G, FENTON W, KIDD K K. Biphasic amplification of very dilute DNA samples via ‘booster’ PCR[J]. Nucleic Acids Research, 1989, 17(13):5407.
[25] ZBRUN M V, MORENO N, CAMUSSONE C M, et al. Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples[J]. Brazilian Journal of Microbiology, 2024, 55(2):1783-1791.
