| 孫晶晶，高曉建，張曉君，馬麗娜，閻斌倫，白雪松，趙佳銘，畢可然，秦蕾.病原性鰻弧菌（Vibrio anguillarum）雙重PCR與LAMP檢測方法的建立.漁業科學進展，2015,36（6）：49-55 |
| 病原性鰻弧菌（Vibrio anguillarum）雙重PCR與LAMP檢測方法的建立 |
| Detection of Pathogenic Vibrio anguillarum by Using Duplex PCR and LAMP Assays |
| 投稿時間：2014-12-16 修訂日期：2015-01-11 |
| DOI：10.11758/yykxjz.20150608 |
| 中文關鍵字： 鰻弧菌 毒力相關基因 雙重PCR 環介導恒溫擴增科技（LAMP） |
| 英文關鍵字： Vibrio anguillarum Virulence gene Duplex PCR LAMP |
| 基金項目：江蘇省高校自然科學研究重大專案（14KJA240001）、江蘇省水產三項工程項目（Y2014-35；D2013-5-4）、江蘇高校優勢學科建設工程項目（2014）和教育部留學回國人員科研啟動基金共同資助 |
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| 摘要點擊次數： 11494 |
| 全文下載次數： 9232 |
| 中文摘要： |
| 本研究檢測了分離自發病大菱鮃、半滑舌鰨及鯉魚的22株病原鰻弧菌（Vibrio anguillarum）毒力相關基因的攜帶情况，並建立了病原鰻弧菌的分子生物學檢測方法。以PCR方法檢測8個毒力相關基因的分佈，結果顯示，22株病原鰻弧菌均可擴增出6個基因（empA、vah1、vah4、flaA、rtxA和tonB）目的條帶，未擴增出virA和angM基因；針對vah4和rtxA設計引子進行雙重PCR擴增，同一PCR反應體系可擴增出兩條目的條帶，靈敏度為2.4×103 CFU/ml，對照菌無任何擴增條帶；以vah4設計引子進行LAMP擴增，病原鰻弧菌可擴增出階梯狀條帶，呈現陽性反應，6株對照菌無階梯狀擴增條帶且呈現陰性反應，LAMP擴增靈敏度為2.4×101 CFU/ml。LAMP檢測靈敏度是雙重PCR的100倍，LAMP科技與PCR比較，操作簡便、快速、靈敏度高且不需昂貴儀器，LAMP檢測鰻弧菌的方法更適合於養殖生產實際應用。 |
| 英文摘要： |
| Vibrio anguillarum widely exists in aquatic environments and has been recognized as one of the prominent waterborne pathogens that undermine the aquaculture industry worldwide.In this study，we investigated the prevalent distribution of eight virulence-associated genes in the V. anguillarum strains isolated from Scophthalmus maximus，Cynoglossus semilaevis and Cyprinus carpio，and improved the detection of V. anguillarum by using duplex PCR and LAMP assays.Six genes（empA，vah1，vah4，flaA，rtxA，and tonB）were detected in all 22 pathogenic strains of V. anguillarum，but virA and angM were not detected.The duplex PCR assay was established with vah4 and rtxA genes as molecular markers.Two gene fragments from the chromosomal DNA of V. anguillarum were detected in one PCR reaction with the detection limit of 2.4×103 CFU/ml. The assay in 6 other control strains generated negative results.The LAMP assay was established with vah4 as the molecular marker.The positive reaction was shown as stair-step amplified bands and the detection limit was 2.4×101 CFU/ml. The assay produced negative reactions in 6 types of control pathogenic bacteria（no amplified bands）.The LAMP method was 100 times more sensitive than the PCR method.Therefore we concluded that the LAMP assay could be a sensitive，rapid and simple tool for the detection of V. anguillarum，and recommend to employ this method in the early diagnosis of V. anguillarum infection in aquatic animals. |
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