| New-type duck reovirus disease(NARV)was a new disease characterized by irregular liver necrosis,plaque hemorrhage,myocardial and bursa of fabricius bleeding.The incidence rate and mortality rate of the disease has large difference,while the smaller the age of diseased ducks is,the higher the incidence rate and mortality rate are.Since 2005,the disease spread quickly from Fujian,Guangdong,Zhejiang to other regions leading to huge economic losses in duck industry.Loop-mediated isothermal amplification(LAMP)was designed for 4 to 6 specific primers in 6 to 8 specific regions of the target gene,and the strand displacement amplification reaction was carried out under isothermal conditions for 30 to 60 min with Bst polymerase.RT-LAMP can complete all the reactions in one step after adding reverse transcriptase based on LAMP.RT-LAMP was strong specificity,high sensitivity,fastness and low equipment requirement.Focusing on the progress of NDRV detection technology and clinical testing needs and taking NDRV-QY as the research object,we used LAMP primer designing software Primer-BLAST,Oligo7.0 and Larsergene7.0 PrimerSelect online to design and primer screening according to the NDRV Sigma B protein gene sequence registered in GenBank.Sigma B gene in virus containing encoding sigma B protein was obtained by PCR and connected to PMD18-T vector to construct recombinant plasmid,which was 1 104 bp.We optimized the reaction conditions to establish NDRV visual RT-LAMP rapid detection method by combining the RT-LAMP technology with fluorescent dye calcein combination and using 6 specific primers in this study.At the same time,We established NDRV RT-LAMP nucleic acid strip rapid detection method by combining RT-LAMP technology with nucleic acid strip technology.Suspected clinical materials were detected by two methods.The results showed that the detection methods established were specific and completed in 50 min under 65 ℃,which could detect the NDRV RNA and the positive plasmid in 200 fglevel,100 times more sensitive than the traditional RT-PCR.The detection rate of methods established and the positive rate of RT-PCR detection was the same in the detection of 15 mixed disease samples,Whlie the former was higher in detecting 12 clinical suspected pathogens.The Positive detection rate of NDRV allantoic fluid in healthy muscovy ducks after Poison attack was 10.71% higher by two methods established than that by theconventional RT-PCR and NDRV can be detected from duck feces in 48 hours after attacking poison.The RT-LAMP calcein visualization method and NDRV-NADA method established aimed at NDRV in this research were greater specificity,higher sensitivity than conventional RT-PCR,which can provide important technical support for the early NDRV diagnosis and monitoring of the new duck reticulosis in grassroots promotion. |
