為建立一種適用于現(xiàn)場(chǎng)快速檢測(cè)的豬流行性腹瀉病毒（PEDV）LAMP技術(shù)，基于羥基萘酚藍(lán)（HNB）的可視化顯色特點(diǎn)，根據(jù)PEDV M基因編碼區(qū)序列，設(shè)計(jì)合成1套引物，通過(guò)反應(yīng)物濃度和反應(yīng)條件優(yōu)化，建立了可閉管檢測(cè)的PEDV RT-LAMP檢測(cè)方法。特異性和靈敏度試驗(yàn)結(jié)果顯示，建立的RT-LAMP檢測(cè)技術(shù)快速、靈敏、特異，可于1 h內(nèi)檢出0.2 mL 0.1 TCID50/mL的病毒RNA，與實(shí)時(shí)熒光RT-PCR檢測(cè)方法靈敏度一致，與豬瘟病毒、豬偽狂犬病病毒、豬繁殖與呼吸綜合征病毒、豬圓環(huán)病毒2型以及豬鏈球菌2型核酸不發(fā)生交叉反應(yīng)。利用該方法對(duì)187份送檢的糞拭子及病死豬組織樣品進(jìn)行應(yīng)用檢測(cè)，檢出陽(yáng)性樣品9份，與熒光定量RT-PCR方法檢測(cè)結(jié)果一致。試驗(yàn)結(jié)果表明，所建立的方法快速、特異，重復(fù)性滿足要求，適用于送檢樣品的PEDV快速檢測(cè)。
Development of HNB-based Visual RT-LAMP Method forDetection
of Porcine Epidemic Diarrhea Virus
In order to establish a RT-LAMP method forrapid detection of porcine epidemic diarrhea virus（PEDV），based on color reaction of HNB and according to M genesequence of PEDV，a set of primers were designed andsynthesized. By optimizing the reaction concentration and conditions，the RT-LAMP method was developed.Specificity and sensitivity results showed the established method was fast，sensitive and specific. It could detectPEDV-RNA extracted from 0.2 mL virus suspension（0.1 TCID50/mL），which was consistent with the real-time RT-PCR method.No cross reactions were observed with the nucleic acids of classical swinefever virus（CSFV），pseudorabies virus（PRV），porcine reproductive and respiratory syndrome virus（PRRSV），porcine circovirus type 2（PCV-2） and Streptococcus suis type 2. Using the RT-LAMPmethod，a total of 9 positive samples were detectedfrom 187 samples of fecal swabs and dead pigs submitted，which was also consistent with the resultsof real-time RT-PCR. As a conclusion，the established method was rapid，specific and repeatable，and it was suitable for rapid detection of PEDV in submitted tissuesamples.
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