重組酶聚合酶擴(kuò)增技術(shù)（recombinase polymerase amplification，RPA ）是近幾年新發(fā)展并普及應(yīng)用的一種核酸擴(kuò)增技術(shù)，能夠?qū)崿F(xiàn)檢測(cè)設(shè)備小型化，從而滿足現(xiàn)場(chǎng)診斷（point-of-care testing，POCT）的需求。本研究以鯉春病毒血癥病毒（spring?viraemia?of?carp virus，SVCV）?為檢測(cè)靶標(biāo)，建立了SVCV的RPA檢測(cè)技術(shù)，并分析了該方法的靈敏性、特異性和穩(wěn)定性，評(píng)判了該技術(shù)應(yīng)用于POCT的可能性。結(jié)果表明：基于RPA的檢測(cè)方法靈敏度較高，檢測(cè)下限可達(dá)4×103 PFU/mL，但比以Taq酶為基礎(chǔ)的RT-PCR技術(shù)略低；特異性好，未發(fā)現(xiàn)對(duì)其他水生動(dòng)物疫病病原，如傳染性造血器官壞死病病毒（IHNV）、病毒性出血性敗血癥病毒（VHSV）、傳染性胰腺壞死病病毒（IPNV）、傳染性鮭魚貧血病病毒（ISAV）等發(fā)生非特異性反應(yīng)；穩(wěn)定性良好，3次重復(fù)性試驗(yàn)的結(jié)果近乎一致。試驗(yàn)證明，該方法能夠替代RT-PCR技術(shù)，應(yīng)用于POCT的試劑和儀器設(shè)備研發(fā)。
Establishment and Evaluation of a Nucleic Acid Detection Technique for Spring Viraemia of Carp Virus Based on RPA
Recombinase polymerase amplification（RPA）is a kind of nucleic acid amplification method developed and widely used in recent years，which could make relevant detection equipments miniaturized to satisfy the requirements of point-of-care testing（POCT）. In this study，based on spring viraemia of carp virus（SVCV），the RPA method for detection of SVCV was established，and its sensitivity，specificity and stability were analyzed，then the possibility of applying this method to POCT was evaluated. The results showed that the method was with high sensitivity，and its lower limit detection was 4×103 PFU/mL which was slightly lower than that of Taq enzyme based RT-PCR；its specificity was good，no any non-specific reaction with the pathogens of other aquatic animal was found，such as IHNV，VHSV，IPNV and ISAV；and the results of three repeated tests were almost the same due to its good stability. In conclusion，the RPA method could be used in research of relevant instruments and reagents for POCT.
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