我国外来入侵生物福寿螺种类的多重PCR鉴别方法
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摘要
为研发能快速、准确地鉴别在我国为害严重的3种福寿螺——小管福寿螺Pomacea canaliculata、斑点福寿螺P. maculata和新发现入侵种Pomacea sp.的PCR技术,基于其线粒体全基因组序列,通过比对分析找到种间差异区段,分别设计并筛选了上述3种福寿螺的特异性引物对,建立了多重PCR检测方法,对其特异性和灵敏度进行评价。结果表明,所设计的引物特异性强,能从不同地理种群的小管福寿螺、斑点福寿螺和Pomacea sp.中分别扩增出1 238、901和571 bp的特异性条带,阴性对照无条带;退火温度为60℃时,32个扩增循环的检测灵敏度可达1 ng样品DNA量。所构建的多重PCR体系可应用于福寿螺不同部位组织碎片、不同性别及不同发育阶段样品的鉴别,具有准确快速、灵敏高效的特点,可用于植保检疫及食品安全部门福寿螺种类的检测。
To develop fast and accurate PCR-based diagnostic methods for three apple snail species, i.e.Pomacea canaliculata, P. maculata and a newly discovered species Pomacea sp., which are damaging crops seriously in China, the species-specific primers for the three species based on the divergent sequences of their whole mitochondrial genomes were designed and screened, and a multiplex PCR was established and the specificity and sensitivity of the primers were assessed. The results showed that the primers were of high specificity, which were able to amplify 1 238, 901, and 571 bp fragments from P.canaliculata, P. maculata, and Pomacea sp., respectively. No amplicon was observed from the negative control. The detective limitation was 1 ng genomic DNA of any of P. canaliculata, P. maculata, and Pomacea sp. in the PCR assay at 60℃ annealing temperature for 32 cycles. This multiplex PCR with species-specific primers could be applied to detecting tissue fragments and samples of both sexes at various developmental stages for assisting species detection of apple snails in plant protection, plant quarantine and food security.
To develop fast and accurate PCR-based diagnostic methods for three apple snail species, i.e.Pomacea canaliculata, P. maculata and a newly discovered species Pomacea sp., which are damaging crops seriously in China, the species-specific primers for the three species based on the divergent sequences of their whole mitochondrial genomes were designed and screened, and a multiplex PCR was established and the specificity and sensitivity of the primers were assessed. The results showed that the primers were of high specificity, which were able to amplify 1 238, 901, and 571 bp fragments from P.canaliculata, P. maculata, and Pomacea sp., respectively. No amplicon was observed from the negative control. The detective limitation was 1 ng genomic DNA of any of P. canaliculata, P. maculata, and Pomacea sp. in the PCR assay at 60℃ annealing temperature for 32 cycles. This multiplex PCR with species-specific primers could be applied to detecting tissue fragments and samples of both sexes at various developmental stages for assisting species detection of apple snails in plant protection, plant quarantine and food security.
引文
Andree K,López MA.2013.Species identification from archived snail shells via genetic analysis:a method for DNA extraction from empty shells.Journal of the Malacological Society of Australia,33(1):1-5
Berthold T.1991.Vergleichende Anatomie,Phylogenie und historische Biogeographie der Ampullariidae(Mollusca:Gastropoda).Abhandlungen des Naturwissenschattlichen Vereinsin Hamburg,29:1-256
Chamberlain JS,Gibbs RA,Ranier JE,Nguyen PN,Caskey CT.1988.Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification.Nucleic Acids Research,16(23):11141-11156
Collier KJ,Demetras NJ,Duggan IC,Johnston TM.2011.Wild record of an apple snail in the Waikato River,Hamilton,New Zealand,and their incidence in freshwater aquaria.New Zealand Natural Sciences,36:1-9
Cooke GM,King AG,Miller L,Johnson RN.2012.A rapid molecular method to detect the invasive golden apple snail Pomacea canaliculata(Lamarck,1822).Conservation Genetics Resources,4(3):591-593
Cowie RH.2002.Apple snails(Ampullariidae)as agricultural pests:their biology,impacts and management.//Barker G.Molluscs as crop pests.Wallingford,UK:CABI Publishing,pp.145-192
Cowie RH,Hayes KA,Thiengo SC.2006.What are apple snails?Confused taxonomy and some preliminary resolution.//Joshi RC,Sebastian LS.Global advances in ecology and management of golden apple snails.Mu?oz,Nueva Ecija:Philippine Rice Research Institute,pp.3-23
Cowie RH,Thiengo SC.2003.The apple snails of the Americas(Mollusca:Gastropoda:Ampullariidae:Asolene,Felipponea,Marisa,Pomacea,Pomella):a nomenclatural and type catalog.Malacologia,45(1):41-100
Dong SZ,Zheng GW,Yu XP,Fu CH.2012.Biological control of golden apple snail,Pomacea canaliculata by Chinese soft-shelled turtle,Pelodiscus sinensis in the wild rice,Zizania latifolia field.Scientia Agricola,69(2):142-146
Gilioli G,Schrader G,Carlsson N,van Donk E,van leeuwen CHA,Martín PR,Pasquali S,VilàM,Vos S.2017.Environmental risk assessment for invasive alien species:a case study of apple snails affecting ecosystem services in Europe.Environmental Impact Assessment Review,65:1-11
Hayes KA,Burks RL,Castro-Vazquez A,Darby PC,Heras H,Martín PR,Qiu JW,Thiengo SC,Vega IA,Wada T,et al.2015.Insights from an integrated view of the biology of apple snails(Caenogastropoda:Ampullariidae).Malacologia,58(1/2):245-302
Hayes KA,Cowie RH,Thiengo SC.2009.A global phylogeny of apple snails:Gondwanan origin,generic relationships,and the influence of outgroup choice(Caenogastropoda:Ampullariidae).Biological Journal of the Linnean Society,98(1):61-76
Hayes KA,Cowie RH,Thiengo SC,Strong EE.2012.Comparing apples with apples:clarifying the identities of two highly invasive Neotropical Ampullariidae(Caenogastropoda).Zoological Journal of the Linnean Society,166(4):723-753
Hu YC,Mu XD,Luo D,Xu M,Yang YX,Gu DG,Luo JR,Zhang JE.2014.Genetic variability of the invasive snail Pomacea canaliculata in South China based on mitochondrial 16S rDNA sequences.Biochemical Systematics&Ecology,57:203-209
Joshi RC,Sebastian LS.2006.Global advances in ecology and management of golden apple snails.Mu?oz,Nueva Ecija:Philippine Rice Research Institute
Kearse M,Moir R,Wilson A,Stones-Havas S,Cheung M,Sturrock S,Buxton S,Cooper A,Markowitz S,Duran C,et al.2012.Geneious Basic:an integrated and extendable desktop software platform for the organization and analysis of sequence data.Bioinformatics,28(12):1647-1649.
Kwong KL,Chan RKY,Qiu JW.2009.The potential of the invasive snail Pomacea canaliculata as a predator of various life-stages of five species of freshwater snails.Malacologia,51(2):343-356
Kwong KL,Dudgeon D,Wong PK,Qiu JW.2010.Secondary production and diet of an invasive snail in freshwater wetlands:implications for resource utilization and competition.Biological Invasions,12(5):1153-1164
Li XY,Bian QQ,Zhao GH.2015.Phylogenetic analysis of Pomacea canaliculata isolates collected from rice fields in different origins of China by combined mitochondrial 12S and 16S genes.Mitochondrial DNA,26(1):27-31
Lowe S,Browne M,Boudjelas S,de Poorter M.2000.100 of the world’s worst invasive alien species:a selection from the global invasive species database.Auckland,New Zealand:Invasive Species Specialist Group of the World Conservation Union
Lv S,Zhang Y,Chen SR,Wang LB,Fang W,Chen F,Jiang JY,Li YL,Du ZW,Zhou XN.2009.Human angiostrongyliasis outbreak in Dali,China.PLoS Neglected Tropical Diseases,3(9):e520
Lv S,Zhang Y,Liu XH,Hu L,Liu Q,Wei FR,Guo YH,Steinmann P,Hu W,Zhou XN,et al.2013.Phylogenetic evidence for multiple and secondary introductions of invasive snails:Pomacea species in the People’s Republic of China.Diversity&Distributions,19(2):147-156
Lv S,Zhang Y,Steinmann P,Yang GJ,Yang K,Zhou XN,Utzinger J.2011.The emergence of angiostrongyliasis in the People’s Republic of China:the interplay between invasive snails,climate change and transmission dynamics.Freshwater Biology,56(4):717-734
Matsukura K,Okuda M,Kubota K,Wada T.2008.Genetic divergence of the genus Pomacea(Gastropoda:Ampullariidae)distributed in Japan,and a simple molecular method to distinguish P.canaliculata and P.insularum.Applied Entomology&Zoology,43(4):535-540
Rawlings TA,Hayes KA,Cowie RH,Collins TM.2007.The identity,distribution,and impacts of non-native apple snails in the continental United States.BMC Evolutionary Biology,7(1):97
Song HM,Hu YC,Wang PX,Mou XD,Li XH,Wang XJ,Luo JR.2010.Sequencing cytochrome oxidase subunit I of mitochondrial DNA and the taxonomic status of apples snails.Chinese Journal of Zoology,45(1):1-7(in Chinese)[宋红梅,胡隐昌,王培欣,牟希东,李小慧,汪学杰,罗建仁.2010.福寿螺线粒体DNA COI基因序列测定及分类地位.动物学杂志,45(1):1-7]
Thaewnon-ngiw B,Klinbunga S,Phanwichien K,Sangduen N,Lauhachinda N,Menasveta P.2004.Genetic diversity and molecular markers in introduced and Thai native apple snails(Pomacea and Pila).Journal of Biochemistry&Molecular Biology,37(4):493-502
Yang QQ,Liu SW,He C,Cowie RH,Yu XP,Hayes KA.2018.Invisible apple snail invasions:importance of continued vigilance and rigorous taxonomic assessments.Pest Management Science,16:doi:10.1002/ps.5241
Yang QQ,Liu SW,Ru WD,Liu GF,Yu XP.2016.Molecular identification of invasive golden apple snails in Zhejiang Province based on DNA barcoding.Biodiversity Science,24(3):341-350(in Chinese)[杨倩倩,刘苏汶,茹炜岽,刘光福,俞晓平.2016.基于DNA条形码技术对浙江省外来入侵福寿螺进行分子鉴定.生物多样性,24(3):341-350]
Yang QQ,Wang ZL,Liu GF,Yu XP.2014.Advance of molecular biology of invasive golden apple snails species in China.Journal of China University of Metrology,25(2):122-128(in Chinese)[杨倩倩,王正亮,刘光富,俞晓平.2014.我国外来入侵种福寿螺分子生物学研究进展.中国计量学院学报,25(2):122-128]
Yoshida K,Matsukura K,Cazzaniga NJ,Wada T.2014.Tolerance to low temperature and desiccation in two invasive apple snails,Pomacea canaliculata and P.maculata(Caenogastropoda:Ampullariidae),collected in their original distribution area(northern and central Argentina).Journal of Molluscan Studies,80(1):62-66
Yu XP,He TJ,Li ZF,LüZX,Sun LP,Zhu YH,Chen JM,Zheng XS,Xu HX.2001.Occurrence of golden apple snails,Pomacea canaliculata(Lamarck),in paddy fields and its management.Acta Agriculturae Zhejiangensis,13(5):247-252(in Chinese)[俞晓平,和田节,李中方,吕仲贤,孙乐平,朱亚红,陈建明,郑许松,徐红星.2001.稻田福寿螺的发生和治理.浙江农业学报,13(5):247-252]
Zhang JE,Zhao BL,Luo MZ,He MQ,Song CX.2010.The ecological risk analysis and assessment on the invasion of golden apple snails.Journal of Foshan University(Natural Science Edition),28(5):1-6(in Chinese)[章家恩,赵本良,罗明珠,何铭谦,宋春秀.2010.外来生物福寿螺入侵的生态风险及其评价探讨.佛山科学技术学院学报(自然科学版),28(5):1-6]
Zhang T,Wang YJ,Guo W,Luo D,Wu Y,Ku?erováZ,Stejskal V,Opit G,Cao Y,Li FJ,et al.2016.DNA barcoding,species-specific PCR and real-time PCR techniques for the identification of six Tribolium pests of stored products.Scientific Reports,6:28494
Zhao ZH,Cui BY,Li ZH,Jiang F,Yang QQ,Ku?erováZ,Stejskal V,Opit G,Cao Y,Li FJ.2016.The establishment of species-specific primers for the molecular identification of ten stored-product psocids based on its2 rDNA.Scientific Reports,6:21022
Zhou XN,Zhang Y,LüS.2009.Proposed Chinese name of Pomacea canaliculata.Chinese Journal of Parasitology and Parasitic Diseases,27(1):62-64(in Chinese)[周晓农,张仪,吕山.2009.“福寿螺”学名中译名的探讨.中国寄生虫学与寄生虫病杂志,27(1):62-64]
Berthold T.1991.Vergleichende Anatomie,Phylogenie und historische Biogeographie der Ampullariidae(Mollusca:Gastropoda).Abhandlungen des Naturwissenschattlichen Vereinsin Hamburg,29:1-256
Chamberlain JS,Gibbs RA,Ranier JE,Nguyen PN,Caskey CT.1988.Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification.Nucleic Acids Research,16(23):11141-11156
Collier KJ,Demetras NJ,Duggan IC,Johnston TM.2011.Wild record of an apple snail in the Waikato River,Hamilton,New Zealand,and their incidence in freshwater aquaria.New Zealand Natural Sciences,36:1-9
Cooke GM,King AG,Miller L,Johnson RN.2012.A rapid molecular method to detect the invasive golden apple snail Pomacea canaliculata(Lamarck,1822).Conservation Genetics Resources,4(3):591-593
Cowie RH.2002.Apple snails(Ampullariidae)as agricultural pests:their biology,impacts and management.//Barker G.Molluscs as crop pests.Wallingford,UK:CABI Publishing,pp.145-192
Cowie RH,Hayes KA,Thiengo SC.2006.What are apple snails?Confused taxonomy and some preliminary resolution.//Joshi RC,Sebastian LS.Global advances in ecology and management of golden apple snails.Mu?oz,Nueva Ecija:Philippine Rice Research Institute,pp.3-23
Cowie RH,Thiengo SC.2003.The apple snails of the Americas(Mollusca:Gastropoda:Ampullariidae:Asolene,Felipponea,Marisa,Pomacea,Pomella):a nomenclatural and type catalog.Malacologia,45(1):41-100
Dong SZ,Zheng GW,Yu XP,Fu CH.2012.Biological control of golden apple snail,Pomacea canaliculata by Chinese soft-shelled turtle,Pelodiscus sinensis in the wild rice,Zizania latifolia field.Scientia Agricola,69(2):142-146
Gilioli G,Schrader G,Carlsson N,van Donk E,van leeuwen CHA,Martín PR,Pasquali S,VilàM,Vos S.2017.Environmental risk assessment for invasive alien species:a case study of apple snails affecting ecosystem services in Europe.Environmental Impact Assessment Review,65:1-11
Hayes KA,Burks RL,Castro-Vazquez A,Darby PC,Heras H,Martín PR,Qiu JW,Thiengo SC,Vega IA,Wada T,et al.2015.Insights from an integrated view of the biology of apple snails(Caenogastropoda:Ampullariidae).Malacologia,58(1/2):245-302
Hayes KA,Cowie RH,Thiengo SC.2009.A global phylogeny of apple snails:Gondwanan origin,generic relationships,and the influence of outgroup choice(Caenogastropoda:Ampullariidae).Biological Journal of the Linnean Society,98(1):61-76
Hayes KA,Cowie RH,Thiengo SC,Strong EE.2012.Comparing apples with apples:clarifying the identities of two highly invasive Neotropical Ampullariidae(Caenogastropoda).Zoological Journal of the Linnean Society,166(4):723-753
Hu YC,Mu XD,Luo D,Xu M,Yang YX,Gu DG,Luo JR,Zhang JE.2014.Genetic variability of the invasive snail Pomacea canaliculata in South China based on mitochondrial 16S rDNA sequences.Biochemical Systematics&Ecology,57:203-209
Joshi RC,Sebastian LS.2006.Global advances in ecology and management of golden apple snails.Mu?oz,Nueva Ecija:Philippine Rice Research Institute
Kearse M,Moir R,Wilson A,Stones-Havas S,Cheung M,Sturrock S,Buxton S,Cooper A,Markowitz S,Duran C,et al.2012.Geneious Basic:an integrated and extendable desktop software platform for the organization and analysis of sequence data.Bioinformatics,28(12):1647-1649.
Kwong KL,Chan RKY,Qiu JW.2009.The potential of the invasive snail Pomacea canaliculata as a predator of various life-stages of five species of freshwater snails.Malacologia,51(2):343-356
Kwong KL,Dudgeon D,Wong PK,Qiu JW.2010.Secondary production and diet of an invasive snail in freshwater wetlands:implications for resource utilization and competition.Biological Invasions,12(5):1153-1164
Li XY,Bian QQ,Zhao GH.2015.Phylogenetic analysis of Pomacea canaliculata isolates collected from rice fields in different origins of China by combined mitochondrial 12S and 16S genes.Mitochondrial DNA,26(1):27-31
Lowe S,Browne M,Boudjelas S,de Poorter M.2000.100 of the world’s worst invasive alien species:a selection from the global invasive species database.Auckland,New Zealand:Invasive Species Specialist Group of the World Conservation Union
Lv S,Zhang Y,Chen SR,Wang LB,Fang W,Chen F,Jiang JY,Li YL,Du ZW,Zhou XN.2009.Human angiostrongyliasis outbreak in Dali,China.PLoS Neglected Tropical Diseases,3(9):e520
Lv S,Zhang Y,Liu XH,Hu L,Liu Q,Wei FR,Guo YH,Steinmann P,Hu W,Zhou XN,et al.2013.Phylogenetic evidence for multiple and secondary introductions of invasive snails:Pomacea species in the People’s Republic of China.Diversity&Distributions,19(2):147-156
Lv S,Zhang Y,Steinmann P,Yang GJ,Yang K,Zhou XN,Utzinger J.2011.The emergence of angiostrongyliasis in the People’s Republic of China:the interplay between invasive snails,climate change and transmission dynamics.Freshwater Biology,56(4):717-734
Matsukura K,Okuda M,Kubota K,Wada T.2008.Genetic divergence of the genus Pomacea(Gastropoda:Ampullariidae)distributed in Japan,and a simple molecular method to distinguish P.canaliculata and P.insularum.Applied Entomology&Zoology,43(4):535-540
Rawlings TA,Hayes KA,Cowie RH,Collins TM.2007.The identity,distribution,and impacts of non-native apple snails in the continental United States.BMC Evolutionary Biology,7(1):97
Song HM,Hu YC,Wang PX,Mou XD,Li XH,Wang XJ,Luo JR.2010.Sequencing cytochrome oxidase subunit I of mitochondrial DNA and the taxonomic status of apples snails.Chinese Journal of Zoology,45(1):1-7(in Chinese)[宋红梅,胡隐昌,王培欣,牟希东,李小慧,汪学杰,罗建仁.2010.福寿螺线粒体DNA COI基因序列测定及分类地位.动物学杂志,45(1):1-7]
Thaewnon-ngiw B,Klinbunga S,Phanwichien K,Sangduen N,Lauhachinda N,Menasveta P.2004.Genetic diversity and molecular markers in introduced and Thai native apple snails(Pomacea and Pila).Journal of Biochemistry&Molecular Biology,37(4):493-502
Yang QQ,Liu SW,He C,Cowie RH,Yu XP,Hayes KA.2018.Invisible apple snail invasions:importance of continued vigilance and rigorous taxonomic assessments.Pest Management Science,16:doi:10.1002/ps.5241
Yang QQ,Liu SW,Ru WD,Liu GF,Yu XP.2016.Molecular identification of invasive golden apple snails in Zhejiang Province based on DNA barcoding.Biodiversity Science,24(3):341-350(in Chinese)[杨倩倩,刘苏汶,茹炜岽,刘光福,俞晓平.2016.基于DNA条形码技术对浙江省外来入侵福寿螺进行分子鉴定.生物多样性,24(3):341-350]
Yang QQ,Wang ZL,Liu GF,Yu XP.2014.Advance of molecular biology of invasive golden apple snails species in China.Journal of China University of Metrology,25(2):122-128(in Chinese)[杨倩倩,王正亮,刘光富,俞晓平.2014.我国外来入侵种福寿螺分子生物学研究进展.中国计量学院学报,25(2):122-128]
Yoshida K,Matsukura K,Cazzaniga NJ,Wada T.2014.Tolerance to low temperature and desiccation in two invasive apple snails,Pomacea canaliculata and P.maculata(Caenogastropoda:Ampullariidae),collected in their original distribution area(northern and central Argentina).Journal of Molluscan Studies,80(1):62-66
Yu XP,He TJ,Li ZF,LüZX,Sun LP,Zhu YH,Chen JM,Zheng XS,Xu HX.2001.Occurrence of golden apple snails,Pomacea canaliculata(Lamarck),in paddy fields and its management.Acta Agriculturae Zhejiangensis,13(5):247-252(in Chinese)[俞晓平,和田节,李中方,吕仲贤,孙乐平,朱亚红,陈建明,郑许松,徐红星.2001.稻田福寿螺的发生和治理.浙江农业学报,13(5):247-252]
Zhang JE,Zhao BL,Luo MZ,He MQ,Song CX.2010.The ecological risk analysis and assessment on the invasion of golden apple snails.Journal of Foshan University(Natural Science Edition),28(5):1-6(in Chinese)[章家恩,赵本良,罗明珠,何铭谦,宋春秀.2010.外来生物福寿螺入侵的生态风险及其评价探讨.佛山科学技术学院学报(自然科学版),28(5):1-6]
Zhang T,Wang YJ,Guo W,Luo D,Wu Y,Ku?erováZ,Stejskal V,Opit G,Cao Y,Li FJ,et al.2016.DNA barcoding,species-specific PCR and real-time PCR techniques for the identification of six Tribolium pests of stored products.Scientific Reports,6:28494
Zhao ZH,Cui BY,Li ZH,Jiang F,Yang QQ,Ku?erováZ,Stejskal V,Opit G,Cao Y,Li FJ.2016.The establishment of species-specific primers for the molecular identification of ten stored-product psocids based on its2 rDNA.Scientific Reports,6:21022
Zhou XN,Zhang Y,LüS.2009.Proposed Chinese name of Pomacea canaliculata.Chinese Journal of Parasitology and Parasitic Diseases,27(1):62-64(in Chinese)[周晓农,张仪,吕山.2009.“福寿螺”学名中译名的探讨.中国寄生虫学与寄生虫病杂志,27(1):62-64]