ZHOU Yanget al: Comparative Study of Nucleic Acid Extraction Method of L.monocytogenes Research Mini-Review Article (Listeria monocytogeneslm) 1

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1 阳等 : 不同食品基质中单核细胞增生李斯特氏菌核酸提取方法的比较阳 12 祝长青 3 郭云昌 4 郭桂萍 3 蒋原 3 刘秀梅 4 付瑞燕 *12 ( ; ; ; ) : 比较 4 类食品基质中单核细胞增生李斯特氏菌核酸提取方法的差异以不同食品基质中的单核细胞增生李斯特氏菌作为研究对象选取应用广泛并且原理不同的 4 种细菌核酸提取方法进行比较同时结合实时荧光 PCR 法对各种方法进行实际评估天根离心柱法核酸提取结果稳定受不同食品基质的影响较小 ;Promega 沉降法与磁珠法受不同食品基质影响较大核酸提取结果差异较大 ;Chelex-100 法快速方便但仅适于纯菌液或菌落的核酸提取 : 单核细胞增生李斯特氏菌 ; 食品基质 ; 核酸提取 ; 实时荧光 PCR : TS :A : (2013) Comparative Study of Nucleic Acid Extraction Method of L.monocytogenes ZHOU Yang 12 ZHU Chang-qing 3 GUO Yun-chang 4 GUO Gui-ping 3 JIANG Yuan 3 LIU Xiu-mei 4 FU Rui-yan *12 (1. School of Tea and Food Science Anhui Agricultural UniversityHefei China;2. Anhui Provincial Key Laboratory of Analysis and Detection Technology for Food SafetyHefei China;3. Jiangsu Entry -Exit Inspection and Quarantine BureauNanjing China;4. National Center for Food Safety Risk Assessment Beijing China) Abstract: Compare the methods of extracting nucleic acid of Listeria monocytogenes in four food matrices. Taking L.monocytogenes as our research objectwe picked and tested four widely applicable nucleic acid extraction methods and evaluated their performance by real -time PCR. Results:Tiangen centrifugal column method was very stablewith minimum variation caused by different food matrices. Promega precipitation and magnetic bead methods were more affected by food matrices than otherswith a larger variation in final products;chelex-100 method was quick and convenientbut was only suitable for pure liquid culture or colonies. Keywords: L.monocytogenesfood matrixthe extraction of nucleic acid real-time fluorescent PCR : : (2012IK168) * : (1976 ) ruiyanfu@189.cn

2 ZHOU Yanget al: Comparative Study of Nucleic Acid Extraction Method of L.monocytogenes Research Mini-Review Article (Listeria monocytogeneslm) 试剂 10 mg/ml; K 20 mg/ml; [1-2] TE :10 mmol/l Tris-HCl (ph 8.0)1 mmol/l EDTA(pH 8.0) ; 75% 50 mmol/l EDTA ; DNA ( ): Cat.#DP302;Promega [3] :Wizard R Genomic DNA Purification KitCat.#A1125;Promega :Wizard R Magnetic DNA Purification Systerm For FoodCat. [4-5] #FF3750;Chelex-100 :5% Chelex % NP40TE ph 8~9; PCR : Cat.#BD-I 仪器 : MB-102; :Thermo Scientific Biofuge Stratos; PCR :Roche LightCycler 480 ; [6] 试验样品 100 g 阴性试验样品的确认 (Polymerase Chain ReactionPCR) GB [8] PCR(real-time PCR) PCR PCR [7] DNA PALCAM DNA 阴性样品增菌培养以及标准菌株增菌液的准备 LB 1 (30±1) 24 h LB 1 1 ml ml LB 2 (30±1) PCR 24 h; CMCC (37±1) 24 h 细菌梯度稀释液的制备 (37±1) 24 h 1 ml 10 ( 实验菌株 ) 100 μl 10-5 ~10-7 ( CMCC 54004: ) (TSA ) (37±1) 培养基及耗材 24 h LB 1 /LB 2 : ; : 样品 ( 阴性增菌液 ) 布菌 1.5 ml TSA OXOID; :TSB 900 μl 100 μl OXOID;PALCAM : OXOID; ( : ;1.5 ml 15 ml ) ( g 10 min) ; 10 ml 2 ml Journal of Food Science and Biotechnology Vol.32 No

3 阳等 : 不同食品基质中单核细胞增生李斯特氏菌核酸提取方法的比较核酸提取 Promega ; Promega ; 2.1 DNA ( ) :100 μl 10 7 ; Chelex-100 [9] 145 : 100 μl Chelex min100 5 min g 2 min 实时荧光 PCR 反应 PCR 10 1 ; μl: 12.5 μl PCR 4 μl PCR 2.2 RT-PCR 3.5 μl 5.0 μl DNA 盐水鸭基质中四种核酸提取方法的比较 :95 10 min1 ;95 10 s58 45 s 50 ;40 40 s1 3 Promega Promega C t PCR ; :100 μl 1 PCR C t 种方法在纯菌核酸提取效果的比较 h Promega ;Promega 100 μl ; ; 1.5 ml Chelex g 10 min 4 Table 1 Fig. 1 1 RT-PCR Real-time PCR amplification curve in salted duck matrix 1 C t C t value of four nucleic acid extraction method in salted duck matrix C t C t

4 ZHOU Yanget al: Comparative Study of Nucleic Acid Extraction Method of L.monocytogenes Research Mini-Review Article 三文鱼基质中 4 种核酸提取方法的比较 2 PCR 3 PCR Promega Promega Promega Promega 3 C t 3 C t Promega Promega ; Promega ;Promega Chelex-100 ; Chelex 牛奶基质中 4 种核酸提取方法的比较 Fig. 2 2 RT-PCR Real-time PCR amplification curve in salmon matrix Table 2 2 C t C t value of four nucleic acid extraction method in salmon matrix C t C t Fig. 3 3 RT-PCR RT-PCR amplification curve in milk matrix 1208 Journal of Food Science and Biotechnology Vol.32 No

5 阳等 : 不同食品基质中单核细胞增生李斯特氏菌核酸提取方法的比较 Table C t C t value of four nucleic acid extraction method in milk matrix C t C t 水果蔬菜色拉基质中 4 种核酸提取方法的 Promega 比较 4 PCR ; 4 ;Promega 10 5 Promega Promega ;Chelex C t Fig. 4 4 RT-PCR Real-time PCR amplification curve in fruit and vegetables salad matrix Table 4 4 C t C t value of four nucleic acid extraction method in fruit and vegetables salad matrix C t C t 纯菌增菌液中四种核酸提取方法的比较 5 Chelex PCR

6 ZHOU Yanget al: Comparative Study of Nucleic Acid Extraction Method of L.monocytogenes Research Mini-Review Article Promega Chelex-100 Fig. 5 5 RT-PCR Real-time PCR amplification curve in pure bacteria liquid Table 5 5 C t C t value of four nucleic acid extraction method in pure bacteria liquid C t C t PCR A D 4 DNA 1210 Journal of Food Science and Biotechnology Vol.32 No

7 阳等 : 不同食品基质中单核细胞增生李斯特氏菌核酸提取方法的比较 DNA PCR ; PCR ; ; DNA ; DNA ;Chelex-100 DNA 4 Chelex-100 DNA : PCR : [ 1 ] Oevermann AZurbriggen AVandevelde M. Rhombencephalitis caused by Listeria monocytogenes in humans and ruminants:a zoonosis on the rise[j]. Interdisciplinary Perspect Infect on Infectious Diseases201011(5): [ 2 ] Freitag N CPort G CMiner M D. Listerial monocytogenes from saprophyte to intracellular pathogen [J]. Nature Reviews Microbiology20107(9): [ 3 ] Jackson K AIwamoto MSwerdlow D. Pregnancy-associated listeriosis[j]. Epidemiol Infect201017:1-7. [ 4 ] Centers for Disease Control and Prevention (CDC). Outbreak of Listeria monocytogenes infections associated with pasteurized milk from a local dairy-massachusetts[j]. Morbidity and Mortality Weekly Report200857(40): [ 5 ] Gilmour M WGraham MDomselaar G Vet al. High -throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborn outbreak[j]. BMC Genomics201011(1): [ 6 ]. PCR [J] (3): WANG NaTAO Yan. Establishment of a multiplex PCR for detection of three types of pathogen in aquatic foods [J]. Journal of Food Science and Biotechnology200928(3): (in Chinese) [ 7 ]. PCR [J] (1): XU Xiao-keWU Qing-pingZHANG Ju-meiet al. Studies on detection of Staphylococus aureus in foods by multiplex PCR[J]. Journal of Food Science and Biotechnology201130(1): (in Chinese) [ 8 ] GB [S]. [ 9 ] Kahankova JSpanova A. Extraction of PCR-ready DNA from Staphylococcus aureus bacteriophages using carboxyl functionalized magnetic Nonporous microspheres[j]. J Chromatogr B Analyt Technol Biomed Life Sci (7):