Development And Application Of The Detection Methods Based On The Recombinase Aided Amplification For DTMUV And A Duplex PCR For DTMUV And N-GPV

Duck Tembusu virus disease(DTMUVD)is an acute,contagious infectious disease caused by duck Tembusu virus(DTMUV).An outbreak of the disease occurred in China in 2010.Initially,DTMUVD mainly infected egg-laying and breeder ducks.Then,it also infected ducklings.The disease can spreads rapidly and widely,therefore,it seriously affected the development of poultry industry in China.Meanwhile,in recent years,a disease,named duck short beak and dwarfism syndrome(DSBDS)characterized by growth retardation with short beaks,protruding and drooping tongues occurred in the commercial broiler duck flocks.A novel goose parvovirus(N-GPV)was identified as the causative agent of the disease.At present,the disease is prevalent in the Cherry Valley duck flocks and clinically mixed infections of the above-mentioned two pathogens are common,which makes the diagnosis and prevention of the disease more difficult.The establishment of novel molecular detection methods that can repid diagnosis of DTMUV infection,as well as quickly identify DTMUV and N-GPV are of great significance for the prevention and control of clinical diseases.Primers and probes were designed synthesized for DTMUV E gene,then a recombinase-aided isothermal amplification assay(RAA)detection technique for rapid detection was established.Meanwhile,primers were designed in this study aims at NS5 gene of DTMUV,VP1 gene of N-GPV,a double PCR detection method for DTMUV and N-GPV was established.The details are as follows:1.Development and application of conventional RAA of DTMUVAccording to the sequences of E gene of DTMUV in Gen Bank,one pair of primers was designed according to the principle of RAA reaction.The reaction conditions were optimized from three aspects of reaction temperature,reaction time and primer concentration.The DTMUV conventional RAA detection method was established and its specificity and sensitivity were analyzed.The specificity results showed that this method could detect DTMUV,while the detection results of NDV,N-GPV,DHAV-1,DHAV-3,DRV,IBV,a MPV,IBDV were negative.The results of sensitivity tests showed that the conventional RAA for DTMUV was determined as 2×10~4copies per reaction,and the sensitivity was the same as that of normal PCR.Nucleic acid amplification could be completed in only 15 min.40 clinical specimens from diseased ducks were used to detect by the established method.The results showed that 22.5%samples were positive,and the results were 100%consistent with normal PCR.2.Development and application of fluorescent RAA of DTMUVAccording to the design principle of fluorescent probe,a probe was designed and a DTMUV fluorescent RAA detection method was established with conventional RAA.Then the specificity and sensitivity was analyzed.The specificity results showed that this method could amplify DTMUV template,while the amplification result of NDV,N-GPV,DHAV-1,DHAV-3,DRV,IBV,a MPV,IBDV were negative.The sensitivity of the method for DTMUV was determined as 2×10~1copies per reaction,and the sensitivity was 1000 times higher than normal PCR and conventional RAA.The fluorescence curve appeared in 5 minutes and the whole amplification and detection process took only 20 minutes.A clinical evaluation of fluorescent recombinase-aided isothermal amplification assay was done using 40 clinical samples.The results showed that 22.5%DTMUV clinical samples were detected,and the results were 100%consistent with normal PCR and conventional RAA.3.Development and application of double RT-PCR for detection of DTMUV and N-GPVBased on the NS5 gene sequence of DTMUV and VP1 gene sequence of N-GPV in Gen Bank,two pairs of specific primers were designed for DTMUV and N-GPV detection.After the optimization of the reaction system and reaction conditions,a double PCR assay was established for the identification of DTMUV and N-GPV,and its specificity,sensitivity and repeatability were analyzed.The results of specificity tests showed that this method could amplify the specific target bands of DTMUV and N-GPV,while the amplification results of NDV,DHAV-1,DHAV-3,DRV,IBV,a MPV,IBDV were negative.Sensitivity tests showed that the minimum detection concentration of the two nucleic acids template by double PCR were 58 fg and 620 fg,respectively.And the minimum detection concentrations of DTMUV and N-GPV by single PCR were 58 fg and62 fg,respectively.The results of repeatability tests showed that the established methods had good repeatability.40 clinical samples were tested by the double PCR for DTMUV and N-GPV,the detection rates of DTMUV and N-GPV were22.5%and 30%,respectively,and the results were 100%consistent with single PCR.In summary,the DTMUV conventional and fluorescence RAA detection method were successfully developed for the frist time,which have the characteristics of good specificity,sensitivity and rapidity,and suitable for rapid on-site detection.Secondly,the double PCR method for DTMUV and N-GPV detection was successfully established for the first time.This method had strong specificity,high sensitivity and high rapidity,providing a new method for rapid diagnosis of the two diseases.