運用微衛星標幟進行台灣豬隻 產銷履歷追溯之驗證

Using microsatellite markers for the traceability test of fresh pork in Taiwan

呂岳錚(Yue-Jheng Lyu)

指導教授 ： 林恩仲

共同指導教授 ： 王佩華(Pei-Hwa Wang)

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.02714

摘要

近年來由於狂牛病的爆發及各種食品安全事件，使得食品安全日漸受到重視，故最近世界各國以歐盟為首，都在推行產銷履歷制度以增進食品安全。傳統使用在動物製品上的產銷履歷以紙本記錄及耳標為主，但是此兩種方法均有造假或錯置的可能，因此利用 DNA 分子標幟進行遺傳追溯對傳統產銷履歷方法進行驗證成為近年來的研究方向之一，由於微衛星標幟具有高多態性、廣泛分布於真核生物染色體及易於使用聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 技術複製等優點，使之常被用來做為遺傳追溯的分子標幟，本研究之目的即在建立可用於台灣地區豬隻產銷履歷驗證之微衛星標幟組。
本研究對來自檢定站，台灣地區常見的三個品種的純種豬隻 (藍瑞斯, 約克夏及杜洛克) 以及來自數個私人豬場之商用雜交肉豬 (藍瑞斯×杜洛克 (LD)，藍瑞斯×約克夏×杜洛克 (LYD) 及未知遺傳背景雜交肉豬 (UnP) 採用10 組微衛星標幟進行研究，計算各標幟之個體鑑別率 (probability of identity, P(ID)) 以確定其個體鑑別之效率，實驗方法為對微衛星標幟引子標記螢光進行兩組多引子PCR 增殖，並進行毛細管電泳以進行基因型鑑定，結果發現三個純品種其理論異質度 (expected heterozyosity, HE) 在無遺傳相關個體上分別為 0.62、0.65 及 0.65；在所有個體間 (部分具有遺傳相關) 分別為 0.61、0.66 及 0.65。其多態性訊息含量 (polymorphism information content, PIC) 在無遺傳相關個體上分別為0.57、0.60 及 0.60；在所有個體間 (部分具有遺傳相關) 分別為 0.57、0.61 及 0.61，三個純品種均在 S0227 有最低的遺傳岐異而在 SW857 有最高的遺傳岐異，進行哈溫平衡 (Hardy-Weinberg equilibrium, HWE) 檢定的結果，在無遺傳相關個體上，藍瑞斯及約克夏各有一個標幟，杜洛克有三個標幟偏離 HWE (P<0.05)，在所有個體間 (部分具有遺傳相關)，藍瑞斯有一個標幟，約克夏及杜洛克各有三個標幟偏離 HWE (P<0.05)，但無論有無遺傳相關，十個標幟之總合均無族群偏離 HWE，表示少數標幟偏離 HWE 對個體鑑別之影響並不顯著。
進行個體鑑定之結果，三個品種其 P(ID) 在遺傳相關個體上分別為 1.42×10-8、6.49×10-9 及 1.23×10-8，在所有個體間 (部分具有遺傳相關) 分別為 1.58×10-8、5.61×10-9 及 9.34×10-9 ，而對雜交豬隻進行個體鑑別之結果，LD 雜交肉豬、LYD 雜交肉豬及 UnP 雜交肉豬其 P(ID) 分別為1.79×10-9、6.14×10-9 及 3.83×10-9，所有族群之 P(ID) 值均小於1×10-7。
另於屠宰場採集民間兩豬場 14 批未知品種肉豬，共 339 頭，與大賣場取得之零售樣品共 8 批進行產銷履歷驗證，可以發現大賣場採得之同批樣品內均有相同之基因型，但僅有一批大賣場樣品可追溯到正確批次之屠宰場採集樣品。其餘之大賣場採樣樣品均未能對應到正確之屠宰場採樣樣品，這些樣品有些具有銷售日期與屠宰日期相差過遠之現象，或是經基因型鑑定的結果，出現了屠宰場採樣樣品所來自之兩豬場所無或極少出現之交替基因。
綜上所述，臺灣近五年之每年上市屠宰的純種淘汰猪及雜交肉猪總數在一千萬頭以下，故屠宰猪在無論有無遺傳相關的情況下，此 10 組微衛星標幟應可達成臺灣常見之純種猪及雜交肉猪的個體鑑別需求，以應用於未來新鮮猪肉產品的產銷履歷回溯驗證。

關鍵字

猪微衛星標幟 ； 產銷履歷 ； 個體鑑別

並列摘要

In the last few decades, Bovine spongiform encephalopathy (BSE) and other food safety issues induced to increase consumer’s concerns of safety in food supply chain. EU member countries and other nations in the world have regulations to ensure food traceability in order to promote food safety. Conventional traceability of animal products consists of labeling system and paper documents, which could be counterfeit and less reliable. To verify the correctness of conventional traceability, genetic traceability based on DNA markers has become popular in recently year. Because microsatellite markers have advantage such as highly polymorphic, abundantly dispersed throughout most eukaryotic nuclear genome, and efficiently amplified using polymerase chain reaction (PCR), they are often utilized as molecular markers in genetic traceability. The objective of this study is to establish the microsatellite marker sets for genetic verification traceability of fresh pork products in Taiwan.
In this study, purebred pigs of three breeds (Landrace, Yorkshire, and Duroc) from the central test station and commercial crossbred pigs (Landrace × Duroc (LD), Landrace × Yorkshire × Duroc (LYD), and unknown genetic background crossbred pigs (UnP) from several private farms were genotyped using ten microsatellite marker sets. The probability of identity (P(ID)) of each marker set in individual genetic traceability was calculated. We performed two multiplex polymerase chain reaction (PCR) with fluorescent-labeled microsatellite markers. The PCR products were then separated by capillary electrophoresis and genotyping. The expected heterozygosity (HE) in none genetic related individuals of three pure breeds were 0.62, 0.65, and 0.65; in all individuals (partial genetic related) of three pure breeds were 0.61, 0.66, and 0.65. The polymorphism information content (PIC) in unrelated individuals of the three breeds were 0.57, 0.60, and 0.60; in all individuals (partial genetic related) of three breeds were 0.57, 0.61, and 0.61. In the three breeds, the genetic diversity was the lowest in the marker of S0227 and the highest in SW857. After performing Hardy-Weinberg equilibrium (HWE) test in the three breeds, in unrelated individuals of the three breeds, there were one in Landrace, one in Yorkshire, and three in Duroc microsatellite markers against HWE respectively (P<0.05), in all individuals (partial genetic related) of three breeds, there were one in Landrace, three in Yorkshire and three in Duroc microsatellite markers against HWE respectively (P<0.05). The combination of observed heterozygosity (HO) with ten microsatellite marker sets showed that the overall situation still agreed with HWE. Therefore, those marker sets against HWE had no significant effects on individual identification.
The results of individual identification showed that P(ID) in unrelated individuals of three breeds were 1.42×10-8, 6.49×10-9, and 1.23×10-8; in all individuals (partial genetic related) of three breeds were 1.58×10-8, 5.61×10-9, and 9.34×10-9. The P(ID) of LD crossbred pigs, LYD crossbred pigs, and UnP were 1.79×10-9, 6.14×10-9, and 3.83×10-9, respectively. All the tested groups had their P(ID) values less than 1.00×10-7.
We collected 339 UnP with 14 batches from two different private farms in slaughter house, and 8 batches of retail sale sample from hypermarkets for traceability test. The result showed that samples from hypermarkets with the same batch number had the same genotypes. Two batches from hypermarkets could trace back to batches from slaughter house correctly. Other samples from hypermarket could not correspond to correct sample collected in slaughter house, some of the samples had issues of the sales date far from slaughtering date, the results of genotyping, the alleles appeared in these samples were rare or even never appeared in these two farms.
In Taiwan, the number of purebred and crossbred pigs slaughtered per year has been less than ten million (1×107) heads in recent five years. Thus, according the results of this study these ten microsatellite markers are sufficient for individual identification among the common purebred and crossbred commercial pigs in the genetic traceability of fresh pork.

並列關鍵字

Pigs ； Microsatellite markers ； Traceability ； Individual identification

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