金黄色葡萄球菌是常见的食源性致病菌之一，近年即食果蔬类食品消费量快速增长，食源性致病菌的控制与检测工作需要快速跟上脚步。实时荧光 PCR 结合高分辨率熔解曲线的技术作为一种分子检测的新方法，在过去十余年中不断发展，在医学领域已取得广泛应用，而在食品致病菌控制领域的运用还不够成熟。因此，针对食源性金黄色葡萄球菌建立一种快速、准确的检测方法，从污染源头控制感染风险十分重要。
本研究开发了一种新型的金黄色葡萄球菌快速选择性增菌培养基，以此为基础使用高分辨率熔解曲线法建立多重实时荧光 PCR 法检测即食果蔬样品中的金黄色葡
萄球菌与其 A 型肠毒素和 U 型肠毒素的靶基因，并将检测体系组建成适用于实际检
测应用的商品化试剂盒。
1. 金黄色葡萄球菌选择性增菌培养基的研制
新型金黄色葡萄球菌选择性增菌培养基（SSA）由葡萄糖和丙酮酸钠作为促进剂，萘啶酮酸和苯乙醇作为抑制剂，加入到 Luria-Bertani 培养基中配制而成。通过响应面实验星点设计法优选出的最佳添加量为葡萄糖 1.73 g/L，丙酮酸钠 12.52 g/L，萘啶酮酸 3.82 mg/L，苯乙醇 1.60 mL/L。SSA 对热损伤金黄色葡萄球菌的恢复率显著优于 LB 与 7.5%氯化钠肉汤。金黄色葡萄球菌在 SSA 中培养的终浓度高于 LB，
生长速度大于 7.5%氯化钠肉汤，而非金黄色葡萄球菌在 SSA 中的生长被抑制。将国
家标准即食果蔬中限量的 10 2 CFU/mL 金黄色葡萄球菌接种到 SSA 培养基中，仅需 6小时培养即可结合分子检测方法得到检测结果。
2. 金黄色葡萄球菌及其肠毒素实时荧光 PCR 法体系的构建
根据实验室筛选的金黄色葡萄球菌特异性靶基因与 A 型和 U 型肠毒素基因设计引物，使用高分辨率熔解曲线结合实时荧光 PCR 的方法建立了一种同时检测金黄色葡萄球菌与其 A 型和 U 型肠毒素的方法。本研究选用 1×的 Syto9 Green 作为荧光染料，实时监测反应的荧光信号值。体系中 Taq 酶浓度为 0.015 U/μL，dNTP 浓度为0.10 mM，终浓度分别为 0.05 μM、0.10 μM 和 0.20 μM 的三对引物扩增出特异性熔解三峰，检测结果清晰。三重检测体系的基因组灵敏度为 4.1 fg/μl，而菌落灵敏度为
1.19×10 3 CFU/mL。金黄色葡萄球菌人工污染即食果蔬，使用 SSA 培养基培养 6 小时，实时荧光 PCR 法检测阳性率为 100%，国标法平板涂布显示检出率则为 90%，高于 7.5%氯化钠肉汤培养 18 小时后 PCR 法 90%与平板涂布法的 40%的检出率。
3. 实时荧光 PCR 试剂盒的构建与应用
将金黄色葡萄球菌及其肠毒素实时荧光 PCR 检测体系构建成试剂盒并加以验证，实现检测方法的商业化应用。试剂盒对各浓度金黄色葡萄球菌的组内和组间重复率均为 100%，在经历 60 次反复冻融，72 小时泡沫箱储存，-20℃储藏 90 天，4℃储藏 45 天后，试剂盒检测结果仍没有显著变化。此外，试剂盒具有良好的特异性与抗干扰能力，对 31 株金黄色葡萄球菌与 15 株非目标菌的检测中均未出现假阴性或假阳性的情况。将 10 6 CFU/mL 的非目标菌与 10 2 CFU/mL 的金黄色葡萄球菌共增菌培养，以及将 100 ng/μL 的非目标菌基因组和 1 ng/μL 的金黄色葡萄球菌基因组共同检测，结果均不受干扰。将试剂盒法与 SSA 培养基相结合，可以提高检测准确性并在 10 小时内得到检测结果。

Staphylococcus aureus is one of the common foodborne pathogens, a rapid growth in consumption of ready-to-eat fruits and vegetables has showed up in the last decade. New method to detect pathogens in these kind of food must be developed quickly. The combination of high-resolution melting curve(HRM) and real-time PCR has provided a new method of molecular detection. In the last few years, HRM has been applied widely in medical domain, however, with the detection of foodborne pathogen, it still had huge
progress to be made. Therefore, this study was aimed to develop a new selective enrichment broth to cultivate the staphylococcus aureus in ready-to-eat fruits and vegetables rapidly, then develop a real-time PCR method combined with HRM for the detection of Staphylococcus aureus and enterotoxin type A and enterotoxin seu, in the end, assemble the components into a kit for commercial sale.
1. The study of selective enrichment broth for Staphylococcus aureus
For the new Staphylococcus aureus selective enrichment broth(SSA), glucose and sodium pyruvate were added into Luria-Bertani as promoters, while nalidixic acid and phenethyl alcohol as inhibitors. Response surface methodology was used to optimize four chosen factors, the optimized concentration were 1.73 g/L of Glucose, 12.52 g/L of sodium pyruvate, 3.82 mg/L of nalidixic acid and 1.60 mL/L of phenethyl alcohol. The growth of S. aureus in SSA was superior to that in LB and faster than TSB with 7.5% NaCl. SSA also showed good inhibition ability against 8 strains which were easy to find in ready-to-eat fruits and vegetables. Further, SSA gave a better recovery environment to heat-stressed pathogen cells than LB and 7.5% NaCl broth. A dosage of 10 2 CFU/mL of S. aureus in fruits and vegetables samples could be detected positive by PCR method after culture of 6 hours by SSA.

2. The establishment of real-time PCR method to detect S. aureus and its enterotoxin
The detection method was established with HRM and real-time PCR. The target gene of Staphylococcus aureus filtrated in our laboratory, enterotoxin type A and enterotoxin seu were used to design specific primers. This study used Syto9 Green with the concentration of 1× as the dye of detection system, which could monitor the signal immediately. The concentration of Ex Taq Hs enzyme and dNTP mixture were 0.015 U/μL and 0.10 mM. Three pairs of primers to amplify three specific resolution peak had concentration of 0.05 μM, 0.10 μM and 0.20 μM. Genome DNA with the concentration of 4.1 fg/μl could be detected with the result of three specific resolution peak, for colony concentration, it came to 1.19×10 3 CFU/mL. After culture of 6 hours in SSA and 18 hours in 7.5% NaCl broth, 10 2 CFU/mL of S. aureus in fruits and vegetables samples were detected by real-time PCR totally and 90% respectively. With baird-parker agar, it only had 90% and 40%.
3. Assemble and application of S. aureus real-time PCR kit
The detection system described above was assembled as a kit and verified to accomplish commercial application. The intra-group and inter-group repeatability of the kit in detection of S. aureus with different concentration were all 100%. The result of kit did not have significant difference after freezing and thawing for 60 times, storing in styrofoam box for 72 hours, 90 days in -20℃ and 45 days in 4℃. Moreover, the kit showed good specificity and capacity of resisting disturbance, no false-positive or false-negative result have been found in detection of 31 S. aureus strains and 15 other strains. In a co-enrichment with other pathogens of 10 6 CFU/mL and S. aureus of 10 2 CFU/mL, there were no significant difference showed up. The same result was achieved in detection of template DNA with 100 ng/μL and S. aureus template DNA with 1 ng/μL. In summary, real-time PCR was demonstrated as a promising rapid kit for the detection of S. aureus and its enterotoxins combined with SSA in a total assay time of 10 h.

[1] 杨丹茹. 金黄色葡萄球菌肠毒素 U 的纯化、检测及其诱发乳腺上皮细胞炎症的初探[D]. 西南

民族大学, 2020.

[2] 索原杰. 多重实时荧光 PCR 致病菌检测方法的构建及其在牛奶中的应用[D]. 浙江大学, 2018.

[3] Kadariya J, Smith T C, Thapaliya D. Staphylococcus aureus and Staphylococcal Food-Borne Disease:

An Ongoing Challenge in Public Health[J]. Biomed Research International, 2014, 2014: 9.

[4] Hennekinne J A, De Buyser M L, Dragacci S. Staphylococcus aureus and its food poisoning toxins:

characterization and outbreak investigation[J]. Fems Microbiology Reviews, 2012, 36(4): 815-836.

[5] Cai H X, Kou X M, Ji H, et al. Prevalence and characteristics of Staphylococcus aureus isolated from

Kazak cheese in Xinjiang, China[J]. Food Control, 2021, 123: 9.

[6] Petroczki F M, Pasztor A, Szucs K D, et al. Occurrence and Characteristics of Staphylococcus aureus

in a Hungarian Dairy Farm during a Control Program[J]. Pathogens, 2021, 10(2): 11.

[7] 杨静. 食源性金黄色葡萄球菌肠毒素基因在液体培养基和牛肉制品中的表达研究[D]. 南京农

业大学, 2013.

[8] Cebrian G, Arroyo C, Manas P, et al. Bacterial maximum non-inhibitory and minimum inhibitory

concentrations of different water activity depressing solutes[J]. International Journal of Food

Microbiology, 2014, 188: 67-74.

[9] Argudin M A, Mendoza M C, Rodicio M R. Food Poisoning and Staphylococcus aureus

Enterotoxins[J]. Toxins, 2010, 2(7): 1751-U342.

[10] Oliveira D, Borges A, Simoes M. Staphylococcus aureus Toxins and Their Molecular Activity in

Infectious Diseases[J]. Toxins, 2018, 10(6): 19.

[11] Otto M. Staphylococcus aureus toxins[J]. Current Opinion in Microbiology, 2014, 17: 32-37.

[12] Suzuki Y. Current Studies of Staphylococcal Food Poisoning[J]. Food Hygiene and Safety Science,

2019, 60(3): 27-37.

[13] Bastos C P, Bassani M T, Mata M M, et al. Prevalence and expression of staphylococcal enterotoxin

genes in Staphylococcus aureus isolated from food poisoning outbreaks[J]. Canadian Journal of

Microbiology, 2017, 63(10): 834-840.

[14] 赵朵, 肖娜, 裴曼君, et al. 金黄色葡萄球菌生物被膜形成及检测方法研究[J]. 教育教学论坛,

2020(47): 388-390.

[15] Archer N K, Mazaitis M J, Costerton J W, et al. Staphylococcus aureus biofilms Properties, regulation

and roles in human disease[J]. Virulence, 2011, 2(5): 445-459.

[16] 马敏. 一起金黄色葡萄球菌肠毒素食物中毒调查分析[J]. 河南预防医学杂志, 2020, 31(12):931-933.

[17] 刘海涛, 吕秋艳, 赵香菊, et al. 一起金黄色葡萄球菌食物中毒事件的实验室溯源分析[J]. 中

国卫生检验杂志, 2020, 30(22): 2793-2795.

[18] 王健. 一起金黄色葡萄球菌引起食物中毒的实验室检测分析[J]. 医学食疗与健康, 2020,

18(18): 13-14.

[19] 陈菲菲, 狄红霞, 蓝乐夫. 金黄色葡萄球菌重要毒力因子的功能及其抑制剂研究进展[J]. 科

学通报, 2013, 58(36): 3743-3752.

[20] 李彦媚, 赵喜红, 徐泽智, et al. 金黄色葡萄球菌引起食物中毒的作用机制与其耐药性的研究

进展[J]. 现代生物医学进展, 2011, 11(14): 2786-2792.

[21] 李毅. 金黄色葡萄球菌及其肠毒素研究进展[J]. 中国卫生检验杂志, 2004(04): 392-395.

[22] 徐振波, 刘晓晨, 李琳, et al. 金黄色葡萄球菌肠毒素在食源性微生物中的研究进展[J]. 现代

食品科技, 2013, 29(09): 2317-2324.

[23] Tarisse C F, Goulard-Huet C, Nia Y, et al. Highly Sensitive and Specific Detection of Staphylococcal

Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay[J]. Toxins, 2021, 13(2): 28.

[24] Alouf J E, Muller-Alouf H. Staphylococcal and streptococcal superantigens: molecular, biological

and clinical aspects[J]. International Journal of Medical Microbiology, 2003, 292(7-8): 429-440.

[25] Ewald S, Notermans S. EFFECT OF WATER ACTIVITY ON GROWTH AND ENTERO-TOXIN

D-PRODUCTION OF STAPHYLOCOCCUS-AUREUS[J]. International Journal of Food

Microbiology, 1988, 6(1): 25-30.

[26] 王璇, 王娉, 葛毅强, et al. 食品中金黄色葡萄球菌致病性研究进展[J]. 中国人兽共患病学报,

2017, 33(06): 553-558.

[27] 向红, 周藜, 廖春, et al. 金黄色葡萄球菌及其引起的食物中毒的研究进展[J]. 中国食品卫生

杂志, 2015, 27(02): 196-199.

[28] Hwang A, Huang L. Ready-to-Eat Foods: Microbial Concerns and Control Measures[M]. 2010.

[29] Balali G I, Yar D D, Afua Dela V G, et al. Microbial Contamination, an Increasing Threat to the

Consumption of Fresh Fruits and Vegetables in Today's World[J]. International Journal of

Microbiology, 2020, 2020: 3029295-Article No.: 3029295.

[30] Hillier-Brown F C, Summerbell C D, Moore H J, et al. A description of interventions promoting

healthier ready-to-eat meals (to eat in, to take away, or to be delivered) sold by specific food outlets

in England: a systematic mapping and evidence synthesis[J]. Bmc Public Health, 2017, 17: 17.

[31] Bari M L, Zaman S: 1 - Microbial biotechnology in food and health: present and future food safety

regulation, Ray R C, editor, Microbial Biotechnology in Food and Health: Academic Press, 2021: 1-

20.[32] Kotzekidou P. Factors influencing microbial safety of ready-to-eat foods[M]. 2016: 33-50.

[33] Yang S, Pei X, Yang D, et al. Microbial contamination in bulk ready-to-eat meat products of China in

2016[J]. Food Control, 2018, 91: 113-122.

[34] Falah M a F, Nadine M D, Suryandono A. Effects of Storage Conditions on Quality and Shelf-life of

Fresh-cut Melon (Cucumis Melo L.) and Papaya (Carica Papaya L.)[J]. Procedia Food Science, 2015,

3: 313-322.

[35] Mahros M M, Abd-Elghany S M, Sallam K I. Multidrug-, methicillin-, and vancomycin-resistant

Staphylococcus aureus isolated from ready-to-eat meat sandwiches: An ongoing food and public

health concern[J]. International Journal of Food Microbiology, 2021: 109165.

[36] Mir S A, Shah M A, Mir M M, et al. Microbiological contamination of ready-to-eat vegetable salads

in developing countries and potential solutions in the supply chain to control microbial pathogens[J].

Food Control, 2018, 85: 235-244.

[37] Huong B T M, Mahmud Z H, Neogi S B, et al. Toxigenicity and genetic diversity of Staphylococcus

aureus isolated from Vietnamese ready-to-eat foods[J]. Food Control, 2010, 21(2): 166-171.

[38] 肖兴宁, 王珍, 蔡铮, et al. 浙江省即食生鲜果蔬病原微生物污染调查分析[J]. 浙江农业科学,

2020, 61(03): 528-530.

[39] 周厚德, 彭思露, 刘道峰, et al. 2015-2017 年江西省部分市售食品中金黄色葡萄球菌的污染情

况调查[J]. 现代预防医学, 2019, 46(12): 2158-2162.

[40] Elias S D O, Decol L T, Tondo E C. Foodborne outbreaks in Brazil associated with fruits and

vegetables: 2008 through 2014[J]. Food Quality and Safety, 2018, 2(4): 173-181.

[41] Jamshidi A, Kazerani H R, Seifi H A, et al. Growth limits of Staphylococcus aureus as a function of

temperature, acetic acid, NaCl concentration, and inoculum level[J]. Iranian Journal of Veterinary

Research, 2008, 9(4): 353-359.

[42] Parfentjev I A, Catelli A R. TOLERANCE OF STAPHYLOCOCCUS AUREUS TO SODIUM

CHLORIDE[J]. Journal of bacteriology, 1964, 88: 1-3.

[43] Hajmeer M, Ceylan E, Marsden J L, et al. Impact of sodium chloride on Escherichia coli O157:H7

and Staphylococcus aureus analysed using transmission electron microscopy[J]. Food Microbiology,

2006, 23(5): 446-452.

[44] Pang L, Luo Y, Gu Y, et al. Recovery Method Development of Sodium Chloride-Susceptible

Methicillin-Resistant Staphylococcus aureus Isolates from Ground Pork Samples[J]. Microbial Drug

Resistance, 2015, 21(1): 1-6.

[45] 李明阳, 胡朋, 高伟敏, et al. 探索 7.5%氯化钠肉汤增菌液 OD 值与金黄色葡萄球菌生物量的

关系[J]. 食品安全质量检测学报, 2019, 10(03): 603-607.[46] 藏程琳. 沙门氏菌快速检测方法构建及致病菌共增菌培养基的研制[D]. 吉林大学, 2020.

[47] 钱红玫. 病原微生物快速检测中的快速增菌方法的优化及其应用研究[D]. 大连工业大学,

2017.

[48] Dodd C E R, Richards P J, Aldsworth T G. Suicide through stress: A bacterial response to sub-lethal

injury in the food environment[J]. International Journal of Food Microbiology, 2007, 120(1-2): 46-

50.

[49] Zhang W L, Jiang W B. UV treatment improved the quality of postharvest fruits and vegetables by

inducing resistance[J]. Trends in Food Science & Technology, 2019, 92: 71-80.

[50] Kabir M N, Aras S, George J, et al. High-pressure and thermal-assisted pasteurization of habituated,

wild-type, and pressure-stressed Listeria monocytogenes, Listeria innocua, and Staphylococcus

aureus[J]. Lwt-Food Science and Technology, 2021, 137: 10.

[51] Lan L, Zhang R, Zhang X, et al. Sublethal injury and recovery of Listeria monocytogenes and

Escherichia coli O157:H7 after exposure to slightly acidic electrolyzed water[J]. Food Control, 2019,

106.

[52] Miller F A, Silva C L M, Brandao T R S. A Review on Ozone-Based Treatments for Fruit and

Vegetables Preservation[J]. Food Engineering Reviews, 2013, 5(2): 77-106.

[53] Lu Y, Turley A, Dong X, et al. Reduction of Salmonella enterica on grape tomatoes using microwave

heating[J]. International Journal of Food Microbiology, 2011, 145(1): 349-352.

[54] Usall J, Ippolito A, Sisquella M, et al. Physical treatments to control postharvest diseases of fresh

fruits and vegetables[J]. Postharvest Biology and Technology, 2016, 122: 30-40.

[55] Fallik E. Prestorage hot water treatments (immersion, rinsing and brushing)[J]. Postharvest Biology

and Technology, 2004, 32(2): 125-134.

[56] Shao L, Liu Y, Tian X, et al. Inactivation and recovery of Staphylococcus aureus in milk, apple juice

and broth treated with ohmic heating[J]. Lwt-Food Science and Technology, 2021, 139.

[57] Bi X, Wang Y, Zhao F, et al. Sublethal injury and recovery of Escherichia coli O157:H7 by high

pressure carbon dioxide[J]. Food Control, 2015, 50: 705-713.

[58] Tian X J, Yu Q Q, Shao L L, et al. Sublethal injury and recovery of Escherichia coli O157:H7 after

ohmic heating[J]. Food Control, 2018, 94: 85-92.

[59] Valderrama W B, Dudley E G, Doores S, et al. Commercially Available Rapid Methods for Detection

of Selected Food-borne Pathogens[J]. Critical Reviews in Food Science and Nutrition, 2016, 56(9):

1519-1531.

[60] 吴任之, 胡欣洁, 韩国全, et al. 食源性金黄色葡萄球菌快速检测方法的研究进展[J]. 食品与

发酵工业: 1-8.[61] Maragos C M: Immunologically-based Methods for Detecting Masked Mycotoxins, Dallasta C,

Berthiller F, editor, Masked Mycotoxins in Food: Formation, Occurrence and Toxicological

Relevance, 2016: 32-49.

[62] Stagnitta P, Micalizzi B, Guzman A M S. Clostridium perfringens enterotoxin quantitative detection

with reversed passive agglutination test (RPLA) in different media[J]. Biocell, 2000, 24(3): 299-299.

[63] Lai Y C, Feldman K L, Clark R S B. Enzyme-linked immunosorbent assays (ELISAs)[J]. Critical

Care Medicine, 2005, 33(12): S433-S434.

[64] Xiang W Q, Peng Z Y, Xu J L, et al. Evaluation of a commercial latex agglutination test for detecting

rotavirus A and human adenovirus in children's stool specimens[J]. Journal of Clinical Laboratory

Analysis, 2020, 34(5): 4.

[65] Rubab M, Shahbaz H M, Olaimat A N, et al. Biosensors for rapid and sensitive detection of

Staphylococcus aureus in food[J]. Biosensors & Bioelectronics, 2018, 105: 49-57.

[66] 刘慧, 曾祥权, 蒋世卫, et al. 生物传感器在食源性金黄色葡萄球菌快速检测中的应用[J]. 食

品科学: 1-18.

[67] Teles F R R, Fonseca L R. Trends in DNA biosensors[J]. Talanta, 2008, 77(2): 606-623.

[68] Epstein J R, Biran I, Walt D R. Fluorescence-based nucleic acid detection and microarrays[J].

Analytica Chimica Acta, 2002, 469(1): 3-36.

[69] Wolcott M J. ADVANCES IN NUCLEIC ACID-BASED DETECTION METHODS[J]. Clinical

Microbiology Reviews, 1992, 5(4): 370-386.

[70] Olsen J E. DNA-based methods for detection of food-borne bacterial pathogens[J]. Food Research

International, 2000, 33(3-4): 257-266.

[71] Souii A, Ben M'hadheb-Gharbi M, Gharbi J. Nucleic acid-based biotechnologies for food-borne

pathogen detection using routine time-intensive culture-based methods and fast molecular

diagnostics[J]. Food Science and Biotechnology, 2016, 25(1): 11-20.

[72] Jofre J, Blanch A R. Feasibility of methods based on nucleic acid amplification techniques to fulfil

the requirements for microbiological analysis of water quality[J]. Journal of Applied Microbiology,

2010, 109(6): 1853-1867.

[73] Sergentet-Thevenot D, Montet M R, Vernozy-Rozand C. Challenges to developing nucleic acid

sequence based amplification technology for the detection of microbial pathogens in food[J]. Revue

De Medecine Veterinaire, 2008, 159(10): 514-527.

[74] Rahman H U, Yue X, Yu Q, et al. Current PCR-based methods for the detection of mycotoxigenic

fungi in complex food and feed matrices[J]. World Mycotoxin Journal, 2020, 13(2): 139-150.

[75] Hanna S E, Connor C J, Wang H H. Real-time polymerase chain reaction for the food microbiologist:Technologies, applications, and limitations[J]. Journal of Food Science, 2005, 70(3): R49-R53.

[76] Gao R, Liao X Y, Zhao X H, et al. The diagnostic tools for viable but nonculturable pathogens in the

food industry: Current status and future prospects[J]. Comprehensive Reviews in Food Science and

Food Safety: 30.

[77] Notomi T, Mori Y, Tomita N, et al. Loop-mediated isothermal amplification (LAMP): principle,

features, and future prospects[J]. Journal of Microbiology, 2015, 53(1): 1-5.

[78] Yang Q R, Domesle K J, Ge B L. Loop-Mediated Isothermal Amplification for Salmonella Detection

in Food and Feed: Current Applications and Future Directions[J]. Foodborne Pathogens and Disease,

2018, 15(6): 309-331.

[79] Kang T S. Basic principles for developing real-time PCR methods used in food analysis: A review[J].

Trends in Food Science & Technology, 2019, 91: 574-585.

[80] Zhang M Y, Ye J, He J S, et al. Visual detection for nucleic acid-based techniques as potential on-site

detection methods. A review[J]. Analytica Chimica Acta, 2020, 1099: 1-15.

[81] Tse C, Capeau J. Real time PCR methodology for quantification of nucleic acids[J]. Annales De

Biologie Clinique, 2003, 61(3): 279-293.

[82] Smith C J, Osborn A M. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches

in microbial ecology[J]. Fems Microbiology Ecology, 2009, 67(1): 6-20.

[83] Hodek J, Ovesna J, Kucera L. Interferences of PCR Effectivity: Importance for Quantitative

Analyses[J]. Czech Journal of Food Sciences, 2009, 27: 42-49.

[84] Taylor S C, Nadeau K, Abbasi M, et al. The Ultimate qPCR Experiment: Producing Publication

Quality, Reproducible Data the First Time[J]. Trends in Biotechnology, 2019, 37(7): 761-774.

[85] Kang S J, Jang C S, Son J M, et al. Comparison of Seven Commercial TaqMan Master Mixes and

Two Real-Time PCR Platforms Regarding the Rapid Detection of Porcine DNA[J]. Food Science of

Animal Resources, 2021, 41(1): 85-94.

[86] Nadkarni M A, Martin F E, Jacques N A, et al. Determination of bacterial load by real-time PCR

using a broad-range (universal) probe and primers set[J]. Microbiology-Sgm, 2002, 148: 257-266.

[87] Osada H, Chon I, Phyu W W, et al. Development of cycling probe based real-time PCR methodology

for influenza A viruses possessing the PA/I38T amino acid substitution associated with reduced

baloxavir susceptibility[J]. Antiviral Research, 2021, 188: 105036.

[88] Livak K J, Flood S J A, Marmaro J, et al. OLIGONUCLEOTIDES WITH FLUORESCENT DYES

AT OPPOSITE ENDS PROVIDE A QUENCHED PROBE SYSTEM USEFUL FOR DETECTING

PCR PRODUCT AND NUCLEIC-ACID HYBRIDIZATION[J]. Pcr-Methods and Applications,

1995, 4(6): 357-362.[89] Liu Y, Gao Y, Wang T, et al. Detection of 12 Common Food-Borne Bacterial Pathogens by TaqMan

Real-Time PCR Using a Single Set of Reaction Conditions[J]. Frontiers in Microbiology, 2019, 10:

9.

[90] Kadiroglu P, Korel F, Ceylan C. Quantification of Staphylococcus aureus in white cheese by the

improved DNA extraction strategy combined with TaqMan and LNA probe-based qPCR[J]. Journal

of Microbiological Methods, 2014, 105: 92-97.

[91] Klotz M, Opper S, Heeg K, et al. Detection of Staphylococcus aureus enterotoxins A to D by real-

time fluorescence PCR assay[J]. Journal of Clinical Microbiology, 2003, 41(10): 4683-4687.

[92] Jansson L, Koliana M, Sidstedt M, et al. Blending DNA binding dyes to improve detection in real-

time PCR[J]. Biotechnology reports (Amsterdam, Netherlands), 2017, 14: 34-37.

[93] Arya M, Shergill I S, Williamson M, et al. Basic principles of real-time quantitative PCR[J]. Expert

Review of Molecular Diagnostics, 2005, 5(2): 209-219.

[94] Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR[J]. Clinical Chemistry and

Laboratory Medicine, 1998, 36(5): 255-269.

[95] Rodriguez-Lazaro D, Hernandez M. Real-time PCR in Food Science: Introduction[J]. Current Issues

in Molecular Biology, 2013, 15(2): 25-37.

[96] Cheng N, Xu Y C, Yan X H, et al. AN ADVANCED VISUAL QUALITATIVE AND EVA GREEN-

BASED QUANTITATIVE ISOTHERMAL AMPLIFICATION METHOD TO DETECT LISTERIA

MONOCYTOGENES[J]. Journal of Food Safety, 2016, 36(2): 237-246.

[97] Pornprasert S, Panyasai S, Waneesorn J, et al. Rapid Identification of Heterozygous and Homozygous

Hemoglobin Constant Spring by SYTO9 with a High Resolution Melting Analysis[J]. Clinical

Laboratory, 2012, 58(7-8): 829-833.

[98] Elkins K M, Perez A C U, Sweetin K C. Rapid and inexpensive species differentiation using a

multiplex real-time polymerase chain reaction high-resolution melt assay[J]. Analytical Biochemistry,

2016, 500: 15-17.

[99] Hasanpour M, Najafi A. Development of a multiplex real-time PCR assay for phylogenetic analysis

of Uropathogenic Escherichia coli[J]. Journal of Microbiological Methods, 2017, 137: 25-29.

[100] Pereira L, Gomes S, Barrias S, et al. Applying high-resolution melting (HRM) technology to olive

oil and wine authenticity[J]. Food Research International, 2018, 103: 170-181.

[101] Rahimi H M, Pourhosseingholi M A, Yadegar A, et al. High-resolution melt curve analysis: A real-

time based multipurpose approach for diagnosis and epidemiological investigations of parasitic

infections[J]. Comparative Immunology Microbiology and Infectious Diseases, 2019, 67: 6.

[102] Asfaram S, Fakhar M, Mirani N, et al. HRM-PCR is an accurate and sensitive technique for thediagnosis of cutaneous leishmaniasis as compared with conventional PCR[J]. Acta Parasitologica,

2020, 65(2): 310-316.

[103] Ebili H, Ilyas M. High Resolution Melt Analysis, DNA Template Quantity Disparities and Result

Reliability[J]. Clinical Laboratory, 2015, 61(1-2): 155-159.

[104] Pakbin B, Basti A A, Khanjari A, et al. Differentiation of stx1A gene for detection of Escherichia

coli serotype O157: H7 and Shigella dysenteriae type 1 in food samples using high resolution melting

curve analysis[J]. Food Science & Nutrition: 8.

[105] Xiao X-L, Zhang L, Wu H, et al. Simultaneous Detection of Salmonella, Listeria monocytogenes,

and Staphylococcus aureus by Multiplex Real-Time PCR Assays Using High-Resolution Melting[J].

Food Analytical Methods, 2014, 7(10): 1960-1972.

[106] Jaeger L H, Nascimento T C, Rocha F D, et al. Adjusting RT-qPCR conditions to avoid unspecific

amplification in SARS-CoV-2 diagnosis[J]. International Journal of Infectious Diseases, 2021, 102:

437-439.

[107] Bustin S A, Nolan T. RT-qPCR Testing of SARS-CoV-2: A Primer[J]. International Journal of

Molecular Sciences, 2020, 21(8): 9.

[108] Bustin S, Huggett J. qPCR primer design revisited[J]. Biomolecular detection and quantification,

2017, 14: 19-28.

[109] Meena B, Anburajan L, Varma K S, et al. A multiplex PCR kit for the detection of three major

virulent genes in Enterococcus faecalis[J]. Journal of Microbiological Methods, 2020, 177: 106061.

[110] Ye L, Zhu Z, Guo Z, et al. Human rare blood group multiplex PCR detection method and kit: US

10266882[P]. Apr 23 2019.

[111] Leamon J, Andersen M, Thornton M. Methods and compositions for multiplex PCR: US

08728728[P]. May 20 2014.

[112] Kawasaki S, Fratamico P M, Kamisaki-Horikoshi N, et al. Development of the Multiplex PCR

Detection Kit for Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7[J]. Jarq-

Japan Agricultural Research Quarterly, 2011, 45(1): 77-81.

[113] Chen W, Li Z G, Yang Y W, et al. Development of the PCR kit for simultaneous detection of

Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus in food[J]. Journal of Food

Agriculture & Environment, 2010, 8(3-4): 96-99.

[114] Lee S H, Jung B Y, Rayamahji N, et al. A multiplex real-time PCR for differential detection and

quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in

meats[J]. Journal of Veterinary Science, 2009, 10(1): 43-51.

[115] Shushe O, Wroblewski D, Macgowan C E, et al. Detection of Salmonella spp. in retail meat products:a comparison between a discontinued commercial kit and a laboratory-developed screening

method[J]. Letters in Applied Microbiology, 2019, 69(2): 116-120.

[116] Duan S Q, Ai J X, Sun L Y, et al. Development and validation of a rapid kit for authenticity of murine

meat in meat products with a species-specific PCR assay[J]. Food Additives and Contaminants Part

a-Chemistry Analysis Control Exposure & Risk Assessment, 2020, 37(4): 552-560.

[117] Zhang R, Lan L S, Shi H. Sublethal injury and recovery of Escherichia coli O157:H7 after freezing

and thawing[J]. Food Control, 2021, 120: 8.

[118] Shao L L, Liu Y, Tian X J, et al. Inactivation of Staphylococcus aureus in phosphate buffered saline

and physiological saline using ohmic heating with different voltage gradient and frequency[J].

Journal of Food Engineering, 2020, 274: 8.

[119] Hu A, Gao C, Lu Z, et al. Detection of Exiguobacterium spp. and E. acetylicum on fresh-cut leafy

vegetables by a multiplex PCR assay[J]. Journal of Microbiological Methods, 2021, 180: 106100.

[120] 陈先锐, 王肇悦, 何秀萍. 酵母菌合成 2-苯乙醇的研究进展[J]. 生物工程学报, 2016, 32(09):

1151-1163.

[121] 朱敏, 梅玲玲, 程苏云. 改良李斯特菌增菌液的研究[J]. 中国卫生检验杂志, 2007(02): 293-

295.

[122] Qu Y, Bai Y L, Liu Y H, et al. SSEL, a selective enrichment broth for simultaneous growth of

Salmonella enterica, Staphylococcus aureus, Escherichia coli O157:H7, and Listeria

monocytogenes[J]. Journal of Food Safety, 2020, 40(5): 13.

[123] 李敏, 马凌云, 陈超阳, et al. 丙酮酸钠药理学作用的研究现状[J]. 中国临床药理学杂志,

2021, 37(03): 341-344.

[124] Yoon J H, Wei S, Oh D H. A highly selective enrichment broth combined with real-time PCR for

detection of Staphylococcus aureus in food samples[J]. Lwt-Food Science and Technology, 2018, 94:

103-110.

[125] Myers R H, Khuri A I, Carter W H. RESPONSE-SURFACE METHODOLOGY - 1966-1988[J].

Technometrics, 1989, 31(2): 137-157.

[126] Lu Y, Turley A, Dong X, et al. Reduction of Salmonella enterica on grape tomatoes using microwave

heating[J]. Int J Food Microbiol, 2011, 145(1): 349-52.

[127] Chen X Q, Tango C N, Daliri E B M, et al. Disinfection Efficacy of Slightly Acidic Electrolyzed

Water Combined with Chemical Treatments on Fresh Fruits at the Industrial Scale[J]. Foods, 2019,

8(10): 19.

[128] Wan J J, Lu Z X, Bie X M, et al. Improvement of a new selective enrichment broth for culturing

Salmonella in ready-to-eat fruits and vegetables[J]. Journal of Food Safety, 2020, 40(5): 12.[129] 郭建平, 万佳佳, 陆兆新, et al. 基于可视化环介导等温扩增技术快速检测金黄色葡萄球菌[J].

食品科学, 2019, 40(20): 325-331.

[130] 张阳. 猪源金黄色葡萄球菌的毒力基因分布，生物被膜形成和凝血致病性的研究[D]. 华南理

工大学, 2018.

[131] 王冬梅, 刘传桂, 梁晓静, et al. 91 株金黄色葡萄球菌毒力基因和耐药基因分布研究[J]. 检验

医学与临床, 2017, 14(14): 2143-2146.

[132] Wang W, Wang X, Wei T, et al. A multiplex real-time PCR approach for identification and

quantification of sheep/goat, fox and murine fractions in meats using nuclear DNA sequences[J].

Food Control, 2021, 126: 108035.

[133] Wang L, Chen F, You D, et al. Development and validation of a multiplex-PCR based assay for the

detection of 18 pathogens in the cerebrospinal fluid of hospitalized children with viral encephalitis[J].

Journal of Virological Methods, 2020, 277: 113804.

[134] Li F, Ye Q, Chen M, et al. Mining of novel target genes through pan-genome analysis for multiplex

PCR differentiation of the major Listeria monocytogenes serotypes[J]. International Journal of Food

Microbiology, 2021, 339: 109026.

[135] Guan H, Xue P, Zhou H, et al. A multiplex PCR assay for the detection of five human pathogenic

Vibrio species and Plesiomonas[J]. Molecular and Cellular Probes, 2021, 55: 101689.

[136] Wolff N, Geiss A F, Bari?i? I. Crosslinking of PCR primers reduces unspecific amplification products

in multiplex PCR[J]. Journal of Microbiological Methods, 2020, 178: 106051.

[137] Zhong Y, Wang Y, Zhao T, et al. Multiplex real-time SYBR Green I PCR assays for simultaneous

detection of 15 common enteric pathogens in stool samples[J]. Molecular and Cellular Probes, 2020,

53: 101619.

[138] Utekal P, Kocanda L, Matousek P, et al. Real-time PCR-based genotyping from whole blood using

Taq DNA polymerase and a buffer supplemented with 1,2-propanediol and trehalose[J]. Journal of

Immunological Methods, 2015, 416: 178-182.

[139] Masalov Y K, Heydarov R N, Shashkov I A, et al. Exogenous contaminating DNA in Taq

polymerases: A method to avoid false-positive results when detecting the blaTEM gene[J]. Journal of

Microbiological Methods, 2019, 160: 36-41.

[140] Masoodi K Z, Lone S M, Rasool R S: Chapter 19 - Polymerase chain reaction (PCR), Masoodi K Z,

Lone S M, Rasool R S, editor, Advanced Methods in Molecular Biology and Biotechnology:

Academic Press, 2021: 109-116.

[141] Liu Z, Sun J, Zhao G, et al. Transient stem-loop structure of nucleic acid template may interfere with

polymerase chain reaction through endonuclease activity of Taq DNA polymerase[J]. Gene, 2021,764: 145095.

[142] Spangler R, Goddard N L, Thaler D S. Optimizing Taq Polymerase Concentration for Improved

Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria[J]. Plos One, 2009, 4(9).

[143] Zhou L, Toydemir R, Wittwer C. Analyzing Copy Number Variation Inheritance with dNTP Limited

PCR and High-resolution Melting Analysis[J]. Journal of Molecular Diagnostics, 2018, 20(6): 1035-

1035.

[144] Wittwer C T, Reed G H, Gundry C N, et al. High-resolution genotyping by amplicon melting analysis

using LCGreen[J]. Clinical Chemistry, 2003, 49(6): 853-860.

[145] Lilliebridge R A, Tong S Y C, Giffard P M, et al. The Utility of High-Resolution Melting Analysis

of SNP Nucleated PCR Amplicons-An MLST Based Staphylococcus aureus Typing Scheme[J]. Plos

One, 2011, 6(6).

[146] Druml B, Cichna-Markl M. High resolution melting (HRM) analysis of DNA – Its role and potential

in food analysis[J]. Food Chemistry, 2014, 158: 245-254.

[147] Farrar J S, Wittwer C T: Chapter 6 - High-Resolution Melting Curve Analysis for Molecular

Diagnostics, Patrinos G P, editor, Molecular Diagnostics (Third Edition): Academic Press, 2017: 79-

102.

[148] Yu Z, Xu Q, Xiao C, et al. SYBR Green real-time qPCR method: Diagnose drowning more rapidly

and accurately[J]. Forensic Science International, 2021, 321: 110720.

[149] Lai M Y, Lau Y L. Detection of Plasmodium knowlesi using recombinase polymerase amplification

(RPA) combined with SYBR Green I[J]. Acta Tropica, 2020, 208: 105511.

[150] Zheng Y, Hu P, Ren H, et al. RPA-SYBR Green I based instrument-free visual detection for

pathogenic Yersinia enterocolitica in meat[J]. Analytical Biochemistry, 2021, 621: 114157.

[151] Monis P T, Giglio S, Saint C P. Comparison of SYTO9 and SYBR Green I for real-time polymerase

chain reaction and investigation of the effect of dye concentration on amplification and DNA melting

curve analysis[J]. Analytical Biochemistry, 2005, 340(1): 24-34.

[152] Forghani F, Singh P, Seo K-H, et al. A novel pentaplex real time (RT)- PCR high resolution melt

curve assay for simultaneous detection of emetic and enterotoxin producing Bacillus cereus in food[J].

Food Control, 2016, 60: 560-568.

[153] 邵晓青, 吕申, 冯璐, et al. 三种荧光染料 SYBR GreenⅠ、LCGreen PLUS、EvaGreen 在实时

定量 PCR 应用中的比较[J]. 大连医科大学学报, 2016, 38(05): 428-431.

[154] Vandesompele J, De Paepe A, Speleman F. Elimination of Primer–Dimer Artifacts and Genomic

Coamplification Using a Two-Step SYBR Green I Real-Time RT-PCR[J]. Analytical Biochemistry,

2002, 303(1): 95-98.[155] Poritz M A, Ririe K M. Getting Things Backwards to Prevent Primer Dimers[J]. The Journal of

Molecular Diagnostics, 2014, 16(2): 159-162.

[156] 邵碧英, 董建, 陈彬, et al. 沙门氏菌核酸检测试剂盒的评价[J]. 食品科学, 2015, 36(24): 242-

245.

[157] Morio F, Poirier P, Le Govic Y, et al. Assessment of the first commercial multiplex PCR kit

(ParaGENIE Crypto-Micro Real-Time PCR) for the detection of Cryptosporidium spp.,

Enterocytozoon bieneusi, and Encephalitozoon intestinalis from fecal samples[J]. Diagnostic

Microbiology and Infectious Disease, 2019, 95(1): 34-37.

[158] Bavisetty S C B, Vu H T K, Benjakul S, et al. Rapid pathogen detection tools in seafood safety[J].

Current Opinion in Food Science, 2018, 20: 92-99.

[159] Ma H, Bell K N, Loker R N. qPCR and qRT-PCR analysis: Regulatory points to consider when

conducting biodistribution and vector shedding studies[J]. Molecular Therapy - Methods & Clinical

Development, 2021, 20: 152-168.

[160] Sena-Torralba A, Pallás-Tamarit Y, Morais S, et al. Recent advances and challenges in food-borne

allergen detection[J]. TrAC Trends in Analytical Chemistry, 2020, 132: 116050.

[161] Van Der Weijden F, Rijnen M, Valkenburg C. Comparison of three qPCR-based commercial tests

for detection of periodontal pathogens[J]. Scientific Reports, 2021, 11(1).

[162] 陈亚明. 奶牛乳房炎致病菌分离鉴定及快速诊断试剂盒的研发与应用[D]. 广西大学, 2014.

[163] 陈思. 牛羊猪犬种布鲁氏菌多重 PCR 方法的建立及试剂盒研制[D]. 中国人民解放军军事医

学科学院, 2014.

[164] 栾云艳. 检测牛奶中布鲁氏菌套式 PCR 试剂盒研制[D]. 黑龙江八一农垦大学, 2016.

[165] Sadykova E, Tyshko N, Grouzdev D, et al. Duplex PCR protocol elaboration for detection and

quantification of GM potato event EH92-527-1 in raw material and food[J]. Febs Open Bio, 2019, 9:

371-372.

[166] Li F, Li B, Dang H, et al. Viable pathogens detection in fresh vegetables by quadruplex PCR[J]. Lwt-

Food Science and Technology, 2017, 81: 306-313.

[167] Gao A L, Fischer-Jenssen J, Cooper C, et al. Evaluation of a Multiplex PCR for Detection of the Top

Seven Shiga-Producing Escherichia coli Serogroups in Ready-to-Eat Meats, Fruits, and Vegetables[J].

Journal of Aoac International, 2018, 101(6): 1828-1832.

[168] Sombat S, Reanwarakorn K, Ling K-S. Developing a multiplex real-time RT-PCR for simultaneous

detection of Pepper chat fruit viroid and Columnea latent viroid[J]. Australasian Plant Pathology,

2018, 47(6): 615-621.

[169] 区楚君, 尚岱麒, 吴酉芝, et al. 消除食品背景杂菌干扰的金黄色葡萄球菌分离方法[J]. 中国食品学报: 1-6.

[170] 周明东. 食源性致病菌快速检测方法的建立及其试剂盒的研制与应用[D]. 山东农业大学,2012.