8 1 Vol. 8 No. 1 2017 1 Journal of Food Safety and Quality Jan., 2017 PCR 万 超 1, 蒋丹 1*, 吕泉 2, 丁健 1, 林长军 1 (1., 116001; 2., 115000) 摘要 : 目的 SYBR Green PCR SYBR Green PCR 方法 CoxⅠ, SYBR Green PCR PCR, DNA DNA 结果 SYBR Green PCR ; 0.06 ng/μl DNA 0.01%, 结论,, 关键词 : ; SYBR Green PCR; PCR identification of Merluccius productus and its products WAN Chao 1, JIANG Dan 1*, LV Quan 2, DING Jian 1, LIN Chang-Jun 1 (1. Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian 116001, China; 2. Bayuquan Entry-Exit Inspection and Quarantine Bureau, Yingkou 115000, China) ABSTRACT: Objective To establish a method for the qualitative detection of Merluccius productus by SYBR Green fluorescence PCR. Methods A set of Merluccius productus specific primers were designed according to the conserve gene of Cox I in Merluccius productus which were amplified by SYBR Green fluorescence PCR method, and the method could be used for the specific detection of DNA from Merluccius productus extracted by DNA kit. Results The primers were highly specific for the detection of Merluccius productus DNA. The limits of detection were 0.06 ng/μl DNA and 0.01% Merluccius productus component. Through the detection of commercially available Pacific hake, Pacific cod and Pacific hake cod liver oil, the component of Merluccius productus in those samples could be detected by this method. Conclusion This method has high specificity and sensitivity, and it can be used for the identification of the component of Merluccius productus in food. KEY WORDS: Merluccius productus; SYBR Green fluorescence PCR; CoxⅠ 基金项目 : (201410059) Fund: Supported by the Commonweal Project Foundation of General Administration of Quality Supervision, Inspection and Quarantine of the People s Republic of China (201410059) * 通讯作者 :,, E-mail: jiangdan66@163.com *Corresponding author: JIANG Dan, Senior Engineer, Liaoning Entry-Exit Inspection and Qurantine Bureau, Dalian 116001, China. E-mail: jiangdan66@163.com
1, : PCR 73 1 Fig. 1 Primer specificity of Merluccius productus 3 结果与分析 3.1 DNA 质量分析 DNA, Nanodrop 2000 260 nm DNA (optical density, OD), DNA DNA 260/280 nm 1.8 2.0, DNA PCR 3.2 引物的特异性 49 DNA, 100 ng DNA 6,, 6 Ct 24.178±0.112, 7 42 ( 2), ;, 49, 3.3 灵敏度检测 5 DNA PCR, DNA 0.06 ng/μl, 3, DNA 0.06 ng/μl PCR ( 0.001%~1%), 0.01%~10%,, 0.001%, 0.01%( ), 4 3.4 市售太平洋无须鳕鱼制品检测结果 1,,, PCR DNA, DNA
74 8 17.5 ng/μl ; 21.5 ng/μl ; 30.3 ng/μl ; 40.06 ng/μl ; 50.012 ng/μl ; 6 1amplification result of 7.5 ng/μl; 2amplification result of 1.5 ng/μl; 3amplification result of 0.3 ng/μl; 4amplification result of 0.06 ng/μl; 5 amplification result of 0.012 ng/μl; 6 negative control 3 DNA Fig. 3 Sensitivity detection of Merluccius productus DNA a. ; b. 1: ; 2-50: ( 49 ) a. amplification curve: b. melting curve 1. Merluccius productus; 2-50: other animal or plant samples (other 49 non-merluccius productus animal and plant samples, such as Gadus macrocephalus, Pollachius virens, Gadus morhua, and so on) Fig. 2 2 Detection of specificity of primers for Merluccius productus 1 10% ; 2 1% ; 3 0.1% ; 4 0.01% ; 5 0.001% ; 6 1amplification result of 10% mass fraction; 2amplification result of 1% mass fraction; 3amplification result of 0.1% mass fraction; 4amplification result of 0.01%mass fraction; 5amplification result of 0.001% mass fraction; 6 negative control 4 Fig. 4 Sensitivity detection of Merluccius productus simulated samples 表 1 市售太平洋无须鳕鱼制品的检测结果 Table 1 Detection result of Merluccius productus products % 30 30 100 5 0 0 15 0 0
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