基于核酸适配体的PCR法检测溶藻弧菌及其灭活菌
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摘要
溶藻弧菌(Vibrio alginolyticus)分布广,数量多,发病率高,是水产养殖中常见的条件致病菌,而对溶藻弧菌进行快速准确的识别鉴定是其病害防治的前提和基础。核酸适配体,因为具有较高的亲和特异性,在微生物的识别鉴定方面展现出了巨大的优势。本文利用核酸适配体和适配体筛选产物,通过结合、洗涤、加热分离、PCR扩增以及电泳检测等步骤,对溶藻弧菌进行了检测鉴定。结果表明,适配体和筛选产物都能对溶藻弧菌及其灭活菌进行较好的识别鉴定,适配体筛选产物对溶藻弧菌的检测下限为10~3cfu/mL,而对其灭活菌的检测下限为10~2cfu/mL,适配体对溶藻弧菌及其灭活菌的检测下限都可达到10 cfu/mL。该方法对溶藻弧菌有较好的亲和特异性,并能较好地区分溶藻弧菌与哈维氏弧菌等水产常见病原菌,在水产病害的检测中显示了较好的应用前景。
Vibrio alginolyticus is a common conditional pathogen in aquaculture, which is widely distributed in the world. Rapid detection of the pathogen is necessary for the prevention and control of the diseases caused by the bacterium. Aptamers exhibit great advantages in the detection of microorganisms owing to their high affinities and specificities. In the present study, Vibrio alginolyticus was detected based on its aptamer and its selection products following various steps, such as binding, washing, separation, amplification, and electrophoresis. The results showed that the aptamer and the selection products could superiorly detect V. alginolyticus and its inactivated form.The lowest detection limits were 10 cfu/mL of V. alginolyticus or its inactivated form by the aptamer, and 10~2 cfu/mL of inactivated V. alginolyticus and 10~3 cfu/mL of V. alginolyticus by the selection products. The system of detection had better affinity and specificity for V. alginolyticus and could distinguish V. alginolyticus from other aquatic pathogens such as V. harveyi, and therefore shows good application potential in the detection of pathogens in aquaculture.
Vibrio alginolyticus is a common conditional pathogen in aquaculture, which is widely distributed in the world. Rapid detection of the pathogen is necessary for the prevention and control of the diseases caused by the bacterium. Aptamers exhibit great advantages in the detection of microorganisms owing to their high affinities and specificities. In the present study, Vibrio alginolyticus was detected based on its aptamer and its selection products following various steps, such as binding, washing, separation, amplification, and electrophoresis. The results showed that the aptamer and the selection products could superiorly detect V. alginolyticus and its inactivated form.The lowest detection limits were 10 cfu/mL of V. alginolyticus or its inactivated form by the aptamer, and 10~2 cfu/mL of inactivated V. alginolyticus and 10~3 cfu/mL of V. alginolyticus by the selection products. The system of detection had better affinity and specificity for V. alginolyticus and could distinguish V. alginolyticus from other aquatic pathogens such as V. harveyi, and therefore shows good application potential in the detection of pathogens in aquaculture.
引文
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[22]Xu Y G,Sun L M,Wang Y S,et al.Simultaneous detection of Vibrio cholerae,Vibrio alginolyticus,Vibrio parahaemolyticus and Vibrio vulnificus in seafood using dual priming oligonucleotide(DPO)system-based multiplex PCR assay[J].Food Control,2017,71:64-70.
[23]Xie Z Y,Pang X H,Zhou Y C,et al.Establishment of a method for detection of Vibrio alginolyticus virulent strains based on a new gene[J].Fisheries Science,2011,30(6):342-346.[谢珍玉,庞兴红,周永灿,等.基于一种新基因的溶藻弧菌毒力菌株检测方法的建立[J].水产科学,2011,30(6):342-346.]
[2]Ye J,Ma Y,Liu Q,et al.Regulation of Vibrio alginolyticus virulence by the LuxS quorum-sensing system[J].Journal of Fish Diseases,2008,31(3):161-169.
[3]Zhao J J,Chen C,Luo P,et al.SYBR Green I-based real-time PCR targeting the rpo X gene for sensitive and rapid detection of Vibrio alginolyticus[J].Molecular and Cellular Probes,2011,25(2-3):137-141.
[4]Hormansdorfer S,Wentges H,Neugebaur-Buchler K,et al.Isolation of Vibrio alginolyticus from seawater aquaria[J].International Journal of Hygiene and Environmental Health,2000,203(2):169-175.
[5]Austin B.Vibrios as causal agents of zoonoses[J].Veterinary Microbiology,2010,140(3-4):310-317.
[6]Sch?rer K,Savioz S,Cernela N,et al.Occurrence of Vibrio spp.in fish and shellfish collected from the Swiss market[J].Journal of Food Protection,2011,74(8):1345-1347.
[7]Schets F M,van den Berg H H J L,Marchese A,et al.Potentially human pathogenic vibrios in marine and fresh bathing waters related to environmental conditions and disease outcome[J].International Journal of Hygiene and Environmental Health,2011,214(5):399-406.
[8]Liu Y,Kong F D,Xu S F,et al.Development and preliminary application of dual PCR assay for Vibrio parahaemolyticus and Vibrio alginolyticus[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(8):647-650.[刘阳,孔繁德,徐淑菲,等.副溶血弧菌和溶藻弧菌双重PCR检测方法的建立与初步应用[J].中国预防兽医学报,2012,34(8):647-650.]
[9]Sawabe T,Kita-Tsukamoto K,Thompson F L.Inferring the evolutionary history of vibrios by means of multilocus sequence analysis[J].Journal of Bacteriology,2007,189(21):7932-7936.
[10]Wei S,Zhao H,Xian Y Y,et al.Multiplex PCR assays for the detection of Vibrio alginolyticus,Vibrio parahaemolyticus,Vibrio vulnificus,and Vibrio cholerae with an internal amplification control[J].Diagnostic Microbiology and Infectious Disease,2014,79(2):115-118.
[11]Li Y N,Zhao J,Zhang A Z,et al.Recent progress in in vitro screening of aptamers[J].Biotechnology Bulletin,2017,33(4):78-82.[李亚楠,赵洁,张傲哲,等.核酸适配体的体外筛选方法的最新研究进展[J].生物技术通报,2017,33(4):78-82.]
[12]Kang K N,Lee Y S.RNA aptamers:a review of recent trends and applications[M]//Future Trends in Biotechnology.Advances in Biochemical Engineering/Biotechnology,2012,131:153-169.
[13]Ding F,Gao Y G,He X R.Recent progresses in biomedical applications of aptamer-functionalized systems[J].Bioorganic&Medicinal Chemistry Letters,2017,27(18):4256-4269.
[14]Kim Y S,Hyun C J,Kim I A,et al.Isolation and characterization of enantioselective DNA aptamers for ibuprofen[J].Bioorganic&Medicinal Chemistry,2010,18(10):3467-3473.
[15]Wang L,Ye H,Sang H Q,et al.Determination of isocarbophos and profenofos B by fluorescent spectrophotometry based on aptamer[J].Chinese Journal of Analytic Chemistry,2016,44(5):799-803.[王丽,叶华,桑宏庆,等.基于核酸适配体的荧光法检测水胺硫磷和丙溴磷[J].分析化学,2016,44(5):799-803.]
[16]Wang L,Wang M K,Shi F P,et al.Aptamer based fluorescence biosensor for protein kinase activity detection and inhibitor screening[J].Sensors and Actuators B:Chemical,2017,252:209-214.
[17]Bayra?C,Eyido?an F,Avni?ktem H.DNA aptamer-based colorimetric detection platform for Salmonella enteritidis[J].Biosensors and Bioelectronics,2017,98:22-28.
[18]Tang X M,Zheng J,Yan Q P,et al.Selection of aptamers against inactive Vibrio alginolyticus and application in a qualitative detection assay[J].Biotechnology Letters,2013,35(6):909-914.
[19]Duan N,Shen M F,Wu S J,et al.Graphene oxide wrapped Fe3O4@Au nanostructures as substrates for aptamer-based detection of Vibrio parahaemolyticus by surface-enhanced Raman spectroscopy[J].Microchimica Acta,2017,184(8):2653-2660.
[20]Duan N,Wu S J,Dai S L,et al.Simultaneous detection of pathogenic bacteria using an aptamer based biosensor and dual fluorescence resonance energy transfer from quantum dots to carbon nanoparticles[J].Microchimica Acta,2015,182(5-6):917-923.
[21]Zhao G T,Ding J W,Yu H,et al.Potentiometric aptasensing of Vibrio alginolyticus based on DNA nanostructure-modified magnetic beads[J].Sensors,2016,16(12):2052.
[22]Xu Y G,Sun L M,Wang Y S,et al.Simultaneous detection of Vibrio cholerae,Vibrio alginolyticus,Vibrio parahaemolyticus and Vibrio vulnificus in seafood using dual priming oligonucleotide(DPO)system-based multiplex PCR assay[J].Food Control,2017,71:64-70.
[23]Xie Z Y,Pang X H,Zhou Y C,et al.Establishment of a method for detection of Vibrio alginolyticus virulent strains based on a new gene[J].Fisheries Science,2011,30(6):342-346.[谢珍玉,庞兴红,周永灿,等.基于一种新基因的溶藻弧菌毒力菌株检测方法的建立[J].水产科学,2011,30(6):342-346.]