论文总字数：20668字

摘 要

本研究建立了普通PCR体系及荧光PCR体系，实现对阿根廷滑柔鱼这一品种鱿鱼的的分子鉴定。利用GenBank查找阿根廷滑柔鱼及多种常见鱿鱼的细胞色素c氧化酶亚基I (Cytochrome Oxidase Subunit I,COI)的核苷酸序列进行对比，设计出特异性引物，并建立验证阿根廷滑柔鱼的PCR体系及荧光PCR体系。在此基础上加入一对已验证的太平洋褶柔鱼特异性引物，建立起阿根廷滑柔鱼与太平洋褶柔鱼的双重PCR及双重荧光PCR鉴定方法。在双重荧光PCR体系中，根据阿根廷滑柔鱼与太平洋褶柔鱼的熔解曲线可与其他物种进行区分。以达到同时对阿根廷滑柔鱼和太平洋褶柔鱼的鲜制品及加工制品准确的分子鉴定。

关键词：阿根廷滑柔鱼 品种鉴定 实时荧光PCR 多重PCR

Establishment and application of SYBR Green I real-time PCR method for detecting Illex argentinus

ABSTRACT

In this study, a common PCR system and a fluorescence PCR system were established to identify the molecular components of the species of squidus argentinea. GenBank was used to find the nucleotide sequences of cytochrome c oxidase subunit I of argentinean leptospira and a variety of common squid for comparison, and specific primers were designed, and the PCR system and fluorescence PCR system for the validation of argentinean leptospira were established. On this basis, a pair of confirmed Pacific pleura specific primers were added to establish a double PCR and double fluorescence PCR identification method for argentinean pleura and Pacific pleura. In the dual fluorescence PCR system, the melt-off curves of the argentinean and Pacific pleated fish can be used to distinguish them from other species. In order to achieve the accurate molecular identification of the fresh and processed products of both argentinean and Pacific pleated fish.

Key Words： Illex argentinus；cultivar identification；Real-time fluorescent PCR ；multiple PCR

目 录

摘要…………………………………………………………………………………I

ABSTRACT………………………………………………………………………II

第一章 文献综述 1

1.1 阿根廷滑柔鱼（Illex argentinus） 1

1.2 水产品品种掺假 2

1.3 基于DNA的检测方法 3

1.3.1 DNA条形码........................................................................................... 3

1.3.2 多重PCR技术...................................................................................... 4

1.3.3 实时荧光PCR技术.............................................................................. 5

1.4目的和意义 5

第二章 材料与方法 6

2.1 样品采集 6

2.2 主要仪器 7

2.3 主要试剂 8

2.4 实验方法 9

2.4.1 DNA提取............................................................................................. 10

2.4.2引物设计与优化.................................................................................. 10

2.4.3 普通PCR.......................................................... .................................. 11

2.4.4 荧光PCR............................................................................................. 12

第三章 实验结果与讨论 12

3.1 DNA 提取 12

3.2 引物退火温度优化及特异性验证 12

3.2.1 引物退火温度优化 12

3.2.2 引物特异性验证 13

3.3 双重普通PCR 14

3.3.1特异性验证 14

3.3.2市售验证 15

3.4 双重荧光PCR 15

3.4.1特异性验证 15

3.3.2市售验证 16

第四章 结论与展望 17

4.1结论 17

4.2展望 17

参考文献 18

致谢 20

1. 文献综述

1.1 阿根廷滑柔鱼（ Illex argentinus）

鱿鱼（Squid），别名枪乌贼、柔鱼，是软体动物门（Mollusca）头足纲（Cephalopoda）蛸亚纲（Coleoidea）十腕总目（Decabrachia）的枪形目（Teuthoidea）的动物^[1]。广义上，鱿鱼是指枪形目中2个亚目、26科、82属、近300个品种的统称^[2-4]，而狭义上的鱿鱼通常是开眼亚目（Oegopsida）中的柔鱼科（Ommastrephidae）品种和闭眼亚目（Myopsina）中的枪乌贼科（Loliginidae）品种的俗称^[1,5]。

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