狂犬病毒、偽狂犬病毒、犬瘟熱病毒、犬細(xì)小病毒和犬冠狀病毒是引起犬科動(dòng)物重要傳染病的5種病毒，嚴(yán)重威脅著犬科動(dòng)物的健康，是出入境犬科動(dòng)物重點(diǎn)篩查的致病因子。為提升口岸進(jìn)出境犬科動(dòng)物檢疫工作效率，建立了上述5種病毒微流控芯片檢測(cè)方法。在建立5種病毒環(huán)等溫?cái)U(kuò)增檢測(cè)技術(shù)（loop mediated isothermal amplification，LAMP）的基礎(chǔ)上，將這些LAMP檢測(cè)體系固化在同一塊塑料芯片上，制備了同步高通量快速檢測(cè)微流控芯片，并將其應(yīng)用于臨床檢測(cè)。結(jié)果顯示：微流控芯片檢測(cè)方法具有良好的特異性，含有目的基因的陽(yáng)性樣本僅在芯片上相應(yīng)病毒反應(yīng)槽出現(xiàn)顯著擴(kuò)增，而其他病毒反應(yīng)槽未出現(xiàn)擴(kuò)增，彼此無(wú)交叉反應(yīng)；微流控芯片的敏感性與LAMP方法一致；通過(guò)檢測(cè)不同時(shí)期收集的感染犬瘟熱病毒、犬細(xì)小病毒和犬冠狀病毒臨床樣本，證實(shí)建立的微流控芯片檢測(cè)方法具有良好的穩(wěn)定性和可靠性。與現(xiàn)有核酸檢測(cè)方法相比，微流控芯片可以在受試核酸上樣40 min內(nèi)完成上述5種犬病毒的快速篩查，從而滿足進(jìn)出境犬科動(dòng)物快速檢疫的需求。
Establishment and Application of the Microfluidic Chip Assay for Five Kinds of Major Canine Viruses
Rabies virus，pseudorabies virus，canine distemper virus，canine parvovirus and canine coronavirus are five kinds of viruses that cause important infectious diseases of canine，which seriously threaten the health of canine，and are the key pathogenic factors to be screened and examined by the exit-entry ports. In order to improve the efficiency of port entry-exit quarantine for canine，a microfluidic chip assay for the above five viruses was established. Five loop-mediated isothermal amplification（LAMP）assays were specifically established and then solidified on the same plastic chip to prepare a microfluidic chip with high-throughput and rapid detection capacity for clinical detection. The results showed that the established assay had a good specificity where positive samples with targeted genes were obviously amplified only in the reaction tank in corresponding viruses instead of other viruses，and no cross reaction with each other was found；the sensitivity of microfluidic chip was consistent with that of LAMP；the established assay was with good stability and reliability as verified through detecting clinical samples infected with canine distemper virus and canine parvovirus and collected in different periods. Compared to existing nucleic acid detection methods，the microfluidic chip could be used to complete rapid screening of the above five viruses within 40 minutes after the loading of nucleic acids，which could satisfy the needs of rapid quarantine for entry-exit canine animals.
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