论文总字数：19071字

摘 要

目前，我国市场沙丁鱼制品种类繁多，传统水产鉴定方法是以形态结构为主。食品加工破坏了沙丁鱼制品的形态学结构特征，使得品种鉴定变的非常困难，从而容易造成沙丁鱼加工制品的品种掺假。DNA条形码为物种的鉴定提供了新的手段，弥补了传统分类的诸多不足。本课题采用通用引物对采集自我国市场的45种常见沙丁鱼制品即22份干制品和23份罐头进行PCR扩增和测序分析。

结果表明，22份干制品和中测序成功的样品有16个，23个罐头中测序成功的有22个。利用BOLD或BLAST数据库序列比对分析发现，测序成功的38个样品可鉴定到属，15个样品可成功鉴定到种，23个样品在物种水平无法进行准确鉴定。鉴定出12个品种，分属11个科。

本研究以中国市售的沙丁鱼资源展开的研究，可以有效地帮助累计基础性研究数据，不断充实数据库，对分类学的发展与微生物资源有着健康积极的推动作用。

关键词: 沙丁鱼 DNA条形码 PCR 序列对比 测序

Application of DNA barcoding for species identification in sardine products

Abstract

At present, there are many kinds of sardine products in China, and the traditional methods for identifying aquatic products are mainly morphological structures. Food processing destroys the morphological structural characteristics of sardine products, making it difficult to identify varieties, which may easily lead to adulteration of sardine processed products. DNA barcodes provide a new means of identifying species and make up for many of the shortcomings of traditional classification. This project used universal primers to perform PCR amplification and sequencing analysis on 45 common sardine products collected from the Chinese market, namely 22 dry products and 23 cans.

The results showed that there were 16 samples of 22 dry products and successful sequencing, and 22 of the 23 cans were successfully sequenced. Using BOLD or BLAST database sequence alignment analysis, it was found that 38 samples successfully sequenced could identify genus, 15 samples could be successfully identified, and 23 samples could not be accurately identified at the species level. Twelve varieties were identified, belonging to 11 families.

This study, based on the research on sardine resources marketed in China, can effectively help to accumulate basic research data, continuously enrich the database, and have a healthy and positive role in the development of taxonomy and microbial resources.

Key words: sardine DNA barcoding PCR Sequence comparison sequencing

目录

摘要 I

Abstract II

第一章 文献综述 1

1.1 沙丁鱼 1

1.1.1 沙丁鱼的生物学分类 1

1.1.2 我国沙丁鱼产业现状 1

1.2 掺假 1

1.2.1 食品掺假 2

1.2.2沙丁鱼掺假 2

1.3 基于DNA的检测方法 3

1.3.1 荧光PCR 3

1.3.2 PCR-FINS技术 3

1.3.3 PCR-RFLP技术 4

1.3.4 DNA条形码及微型DNA条形码 4

1.4 目的及意义 5

第二章 材料与方法 6

2.1 采集样品 6

2.2 主要试剂与仪器 6

2.2.1 主要试剂 6

2.2.2 主要仪器 7

2.3实验方法 8

2.3.1 DNA提取 8

2.3.2 浓度测定 8

2.3.3 总电泳 8

2.3.4 PCR扩增及测序 8

2.5 PCR产物电泳和测序 9

2.3.6 序列比对与分析 9

第三章 结果与讨论 11

3.1 DNA提取 11

3.2 总电泳 11

3.3 PCR扩增与测序 12

3.4序列比对与分析 14

第四章 结论与展望 17

4.1 结论 17

4.2 问题与展望 17

参考文献 19

致谢 22

第一章 文献综述

1.1 沙丁鱼

1.1.1 沙丁鱼的生物学分类

沙丁鱼是一大类硬骨鱼纲（Osteichthyes）鲱形目（Clupeiformes）中食用鱼的统称。联合国粮食及农业组织/世界卫生组织对罐装沙丁鱼的标准引用了21种可归类为沙丁鱼的物种。这21种沙丁鱼为：Sardina pilchardus、Sardinops melanostictus、S. neopilchardus、S. ocellatus、S. sagax、S. caeruleus、Sardinella aurita、S. brasiliensis、S. maderensis、S. longiceps、S. gibbosa、Clupea harengus、Clupea bentincki、Sprattus sprattus、Hyperlophus vittatus、Nematalosa vlaminghi、Etrumeus teres、Ethmidium maculatum、Engraulis anchoita、E. mordax、E. ringens、Opisthonema oglinum^[1]。

1.1.2 我国沙丁鱼产业现状

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