非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)感染家猪和野猪引起的一种急性出血性严重传染病。ASF的持续传播不仅影响到居民的肉类供应,也影响到全球肉类供应的安全,因此,建立灵敏特异的ASFV检测方法已经成为一个食品安全重要热点。利用量子点(quantum dots,QDs)和纳米金(Au nanoparticles,AuNPs)构建了一种基于荧光共振能量转移(fluorescence resonance energy transfer, FRET)的DNA传感器,基于单链DNA互补配对原则用于ASFV特异性基因检测。在靶DNA缺失的情况下,ss-DNA-QDs(探针 1)将与ss′-DNA-AuNPs (探针 2)杂交,供体QDs与受体AuNPs距离变近,引发FRET效应,QDs的荧光被AuNPs淬灭。然而,靶DNA存在的情况下,靶DNA与探针2竞争结合探针1,导致FRET被破坏,QDs的荧光恢复。该生物传感器可在1.25 h内快速检测ASFV靶DNA,检出限为0.72 μmol/L,在猪肉、火腿肠和猪肉饺子等食品中的回收率为82.00%~108.00%,变异系数为0.02%~0.15%。该研究提出了一种简单、快速的检测食品中ASFV基因片段的方法。在以后的研究中,可以通过优化条件来提高灵敏度。此外,这种策略可以扩展到其他DNA、病毒或蛋白质的传感应用。
African swine fever (ASF) is an acute, hemorrhagic and severe infectious disease caused by African swine fever virus (ASFV) which infect domestic pigs and wild boars. The continued spread of ASF not only affects the meat supply of residents, but also the safety of global meat supplied. Therefore, the establishment of sensitive and specific ASFV detection method has become an important issue in food safety. In this work, we reported a fluorescence resonance energy transfer (FRET) DNA biosensor based on quantum dots (QDs) and gold nanoparticles (AuNPs) for ASFV specific gene detection. In the absence of target DNA, ss-DNA-QDs (Probe 1) will hybridize with ss'-DNA-AuNPs (Probe 2) making the donor (QDs) and the acceptor (AuNPs) close to each other and trigger FRET effect, and the fluorescence of QDs was quenched by AuNPs. However, in the presence of target DNA, the target DNA competed with Probe 2 to bind Probe 1, which resulted in the destruction of FRET and recovery of the fluorescence of QDs. The developed biosensor in here could achieve rapid detection of ASFV target DNA with the detection limit of 0.72 μmol/L in 1.25 h. Which was verified in pork, ham sausage, and pork dumplings with recoveries from 82.00% to 108.00% and variation coefficients of 0.02%-0.15%. This study proposed a new, simple and rapid method to detect ASFV gene fragment in food. In addition, this strategy can be used to other DNA, virus or protein sensing applications.
[1] GABRIEL C,BLOME S,MALOGOLOVKIN A,et al.Characterization of African swine fever virus caucasus isolate in European wild boars[J]. Emerging Infectious Diseases, 2011, 17(12):2 342-2 345.
[2] ZAKARYAN H,REVILLA Y.African swine fever virus:Current state and future perspectives in vaccine and antiviral research[J].Veterinary Microbiology,2016,185:15-19.
[3] PENRITH M L,PEREIRA C L,DA SILVA M M R L,et al.African swine fever in Mozambique:Review,risk factors and considerations for control[J].Onderstepoort Journal of Veterinary Research,2007,74(2):149-160.
[4] TUBIASH H S.Quantity production of leukocyte cultures for cultures use in hemadsorption tests with African swine fever virus[J].American Journal of Veterinary Research,1963,24:381-384.
[5] 陈静. 浅谈目前非洲猪瘟常用诊断方法[J].中国畜禽种业,2020,16(10):152-153.
CHEN J.A brief discussion on the common diagnostic methods of African swine fever[J].The Chinese Livestock and Poultry Breeding,2020,16(10):152-153.
[6] GIMENEZ-LIROLA L G,MUR L,RIVERA B,et al.Detection of African swine fever virus antibodies in serum and oral fluid specimens using a recombinant protein 30 (p30) dual matrix indirect ELISA[J].PLoS One,2016,11(9):e0161230.
[7] BERGERON H C,GLAS P S,SCHUMANN K R.Diagnostic specificity of the African swine fever virus antibody detection enzyme-linked immunosorbent assay in feral and domestic pigs in the United States[J].Transboundary and Emerging Diseases,2017,64(6):1 665-1 668.
[8] 张元峰, 刘锡玲,毕路,等.胶体金检测试纸卡在检测非洲猪瘟病毒抗体上的应用[J].养殖与饲料,2020,19(11):15-18.
ZHANG Y F,LIU X L,BI L,et al.Application of colloidal gold test paper card in detection of antibodies against African swine fever virus[J].Animals Breeding and Feed,2020,19(11):15-18.
[9] AGUERO M,FERNANDEZ J,ROMERO L,et al.Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples[J].Journal of Clinical Microbiology,2003,41(9):4 431-4 434.
[10] FERNANDEZ-PINERO J,GALLARDO C,ELIZALDE M,et al.Molecular diagnosis of African swine fever by a new real-time PCR using universal probe library[J].Transboundary and Emerging Diseases,2013,60(1):48-58.
[11] HAINES F J,HOFMANN M A,KING D P,et al.Development and validation of a multiplex,real-time RT PCR assay for the simultaneous detection of classical and African swine fever viruses[J].PLoS One,2013,8(7):e71019.
[12] AGUERO M,FERNANDEZ J,ROMERO L J,et al.A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and classical swine fever in clinical samples[J].Veterinary Research,2004,35(5):551-563.
[13] RONISH B,HAKHVERDYAN M,STAHL K,et al.Design and verification of a highly reliable Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus[J].Journal of Virological Methods,2011,172(1-2):8-15.
[14] JAMES H E,EBERT K,MCGONIGLE R,et al.Detection of African swine fever virus by loop-mediated isothermal amplification[J].Journal of Virological Methods,2010,164(1-2):68-74.
[15] 王林, 高晓龙,吴迪,等.非洲猪瘟病毒实时荧光LAMP检测方法的建立与应用[J].中国兽药杂志,2020,54(8):1-8.
WANG L,GAO X L,WU D,et al.Establishment and application of real-time fluorescent LAMP detection method for African swine fever virus[J].Chinese Journal of Veterinary Drug,2020,54(8):1-8.
[16] WANG J C,WANG J F,GENG Y Y,et al.A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus[J].Canadian Journal of Veterinary Research,2017,81(4):308-312.
[17] FRACZYK M,WOZNIAKOWSKI G,KOWALCZYK A,et al.Development of cross-priming amplification for direct detection of the African swine fever virus,in pig and wild boar blood and sera samples[J].Letters in Applied Microbiology,2016,62(5):386-391.
[18] SINGURU M M R,SUN S C,CHUANG M C.Advances in oligonucleotide-based detection coupled with fluorescence resonance energy transfer[J].Trac-Trends in Analytical Chemistry,2020,123:115 756.
[19] ZHANG X J,HU Y,YANG X T,et al.Förster resonance energy transfer (FRET)-based biosensors for biological applications[J].Biosensors & Bioelectronics,2019,138:111 314.
[20] LI C C,LI Y,ZHANG Y,et al.Single-molecule fluorescence resonance energy transfer and its biomedical applications[J].Trac-Trends in Analytical Chemistry,2020,122:115 753.
[21] JIN B R,WANG S R,LIN M,et al.Upconversion nanoparticles based FRET aptasensor for rapid and ultrasenstive bacteria detection[J].Biosensors & Bioelectronics,2017,90:525-533.
[22] LU M L,MA X C,MOTHES W.Illuminating the virus life cycle with single-molecule FRET imaging[J].Advances in Virus Research,2019,105:239-273.
[23] ZHANG W,LIU X L,LI P,et al.Cellular fluorescence imaging based on resonance energy transfer[J].Trac-Trends in Analytical Chemistry,2020,123:115 742.
[24] HUANG H P,ZHU J J.DNA aptamer-based QDs electrochemiluminescence biosensor for the detection of thrombin[J].Biosensors & Bioelectronics,2009,25(4):927-930.
[25] FRENS G.controlled nucleation for regulation of particle-size in monodisperse gold suspensions[J].Nature Physical Science,1973,241(105):20-22.
[26] LIU J W,LU Y.Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes[J].Nature Protocols,2006,1(1):246-252.
[27] GUO J J,QIU X,MINGOES C,et al.Conformational details of quantum dot-DNA resolved by Förster Resonance Energy Transfer lifetime nanoruler[J].ACS Nano,2019,13(1):505-514.
[28] ZHANG D Y,WINFREE E.Control of DNA strand displacement kinetics using toehold exchange[J].Journal of the American Chemical Society,2009,131(47):17 303-17 314.
[29] WANG D,CHEN H,LI H,et al.Detection of Staphylococcus aureus carrying the gene for toxic shock syndrome toxin 1 by Quantum-Dot-Probe complexes[J].Journal of Fluorescence,2011,21(4):1 525-1 530.
[30] LIU B W,LIU J W.Freezing directed construction of Bio/Nano Interfaces:Rreagentless conjugation,denser spherical nucleic acids,and better nanoflares[J].Journal of the American Chemical Society,2017,139(28):9 471-9 474.
