PMA-PCR方法快速检测VBNC状态青枯菌
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摘要
青枯菌为应对逆境胁迫,可进入活的但非可培养状态(viable but non-culturable,VBNC)。本文利用叠氮溴化丙锭(PMA)与PCR技术相结合,建立了一种快速有效区分青枯菌死活细胞的分子检测方法。基于hrcS基因序列,设计了一对青枯菌种特异性检测引物hrcSf/hrcSr;利用PMA对青枯菌Po82菌株的细胞悬浮液样品进行预处理,随后进行常规PCR扩增。结果表明,当样品中PMA质量浓度为3μg/mL、曝光时间大于5min时,PMA可有效抑制死亡菌体细胞中的DNA扩增;且对可培养和VBNC状态细胞中的DNA扩增没有影响;本试验建立的PMA-PCR方法能有效对包括VBNC状态在内的青枯菌活菌进行检测,避免了假阳性与假阴性结果的产生。
Ralstonia solanacearum can enter into the VBNC state(viable but non-culturable)in response to adverse environmental conditions.A novel method to differentiate viable/dead cells of R.solanacearum was established by using a DNA dye of propidium monoazide(PMA)in combination with the polymerase chain reaction(PMA-PCR).On the basis of hrcS gene sequence,a species-specific primer set hrcSf/hrcSr was designed for detection of R.solanacearum.Samples were pre-treated with PMA which bound DNA from dead cells so that only viable cells can be amplified by PCR.A final concentration of 3μg/mL PMA and 5-min exposure time was found to completely inhibit PCR amplification from DNA of dead cells,and had no inhibition effect on viable and viable but non-culturable(VBNC)cells.The PMA-PCR method established in this work can detect viable and viable but non-culturable(VBNC)cells and avoid false positive and false negation result of R.solanacerum.
Ralstonia solanacearum can enter into the VBNC state(viable but non-culturable)in response to adverse environmental conditions.A novel method to differentiate viable/dead cells of R.solanacearum was established by using a DNA dye of propidium monoazide(PMA)in combination with the polymerase chain reaction(PMA-PCR).On the basis of hrcS gene sequence,a species-specific primer set hrcSf/hrcSr was designed for detection of R.solanacearum.Samples were pre-treated with PMA which bound DNA from dead cells so that only viable cells can be amplified by PCR.A final concentration of 3μg/mL PMA and 5-min exposure time was found to completely inhibit PCR amplification from DNA of dead cells,and had no inhibition effect on viable and viable but non-culturable(VBNC)cells.The PMA-PCR method established in this work can detect viable and viable but non-culturable(VBNC)cells and avoid false positive and false negation result of R.solanacerum.
引文
[1]Schell M A.Control of virulence and pathogenicity genes of Ralstonia solanacearum by an elaborate sensory network[J].Annual Review of Phytopathology,2000,38:263-292.
[2]徐进,冯洁.植物青枯菌遗传多样性及致病基因组学研究进展[J].中国农业科学,2013,46(14):2902-2909.
[3]Genin S,Boucher C.Lessons learned from the genome analysis of Ralstonia solanacearum[J].Annual Review of Phytopathology,2004,42:107-134.
[4]Milling A,Meng Fanhong,Denny T P,et al.Interactions with hosts at cool temperatures,not cold tolerance,explain the unique epidemiology of Ralstonia solanacearumrace 3biovar 2[J].The American Phytopathological Society,2009,99(10):1127-1134.
[5]蒋娜,李健强,罗来鑫.植物病原细菌的VBNC状态研究进展[J].植物病理学报,2013,43(3):249-257.
[6]del Campo R,Russi P,Mara P,et al.Xanthomonas axonopodis pv.citri enters the VBNC state after copper treatment and retains its virulence[J].FEMS Microbiology Letters,2009,298(2):143-148.
[7]Alexander E,Pham D,Steck T R.The viable but nonculturable condition is induced by copper in Agrobacterium tumefaciens and Rhizobium leguminosarum[J].Applied and Environmental Microbiology,1999,65(8):3754-3756.
[8]Grey B E,Steck T R.The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection[J].Applied and Environmental Microbiology,2001,67(9):3866-3872.
[9]潘乐,孟庆峰,钱爱东,等.VBNC状态细菌检测方法的研究进展[J].中国农学通报,2012,28(5):32-35.
[10]Nocker A,Cheung C Y,Camper A K.Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs dead bacteria by selective removal of DNA from dead cells[J].Journal of Microbiological Methods,2006,67(2):310-320.
[11]Wang Luxin,Mustapha A.EMA-real-time PCR as a reliable method for detection of viable salmonella in chicken and eggs[J].Journal of Food Science,2010,75(3):M134-M139.
[12]熊书,殷幼平,王芳,等.基于EMA-qPCR的茄科青枯菌活体检测技术的建立[J].微生物学通报,2013,40(9):1723-1732.
[13]Ramamurthy T,Ghosh A,Pazhani G P,et al.Current perspectives on viable but nonculturable(VBNC)pathogenic bacteria[J].Frontiers in Public Health,2014,2:103.
[14]Trevors J T.Viable but nonculturable(VBNC)bacteria:Gene expression in planktonic and biofilm cells[J].Journal of Microbiological Methods,2011,86(2):266-273.
[15]Oliver J D.The viable but nonculturable state in bacteria[J].The Journal of Microbiology,2005,43:93-100.
[16]Oliver J D,Bockian R.In vivo resuscitation,and virulence towards mice,of viable but nonculturable cells of Vibrio vulnificus[J].Applied and Environment Microbiology,1995,61(7):2620-2623.
[17]Biosecurity Australia.Revised draft import risk analysis report for the importation of cavendish bananas from the philippines,part C[R].Canberra:Biosecurity Australia,2007.
[18]Zanón M J,Font M I,JordáC.Use of tomato crop residues into soil for control of bacterial wilt caused by Ralstonia solanacearum[J].Crop Protection,2011,30(9):1138-1143.
[19]Paret M L.Management of bacterial wilt of ginger(Zingiber officinale R.)caused by Ralstonia solanacearum with plant essential oils[D].Hawaii:University of Hawaii,2009.
[20]Alfano J R,Collmer A.The typeⅢ(Hrp)secretion pathway of plant pathogenic bacteria:trafficking harpins,Avr proteins,and death[J].Journal of Bacteriology,1997,179(18):5655-5662.
[2]徐进,冯洁.植物青枯菌遗传多样性及致病基因组学研究进展[J].中国农业科学,2013,46(14):2902-2909.
[3]Genin S,Boucher C.Lessons learned from the genome analysis of Ralstonia solanacearum[J].Annual Review of Phytopathology,2004,42:107-134.
[4]Milling A,Meng Fanhong,Denny T P,et al.Interactions with hosts at cool temperatures,not cold tolerance,explain the unique epidemiology of Ralstonia solanacearumrace 3biovar 2[J].The American Phytopathological Society,2009,99(10):1127-1134.
[5]蒋娜,李健强,罗来鑫.植物病原细菌的VBNC状态研究进展[J].植物病理学报,2013,43(3):249-257.
[6]del Campo R,Russi P,Mara P,et al.Xanthomonas axonopodis pv.citri enters the VBNC state after copper treatment and retains its virulence[J].FEMS Microbiology Letters,2009,298(2):143-148.
[7]Alexander E,Pham D,Steck T R.The viable but nonculturable condition is induced by copper in Agrobacterium tumefaciens and Rhizobium leguminosarum[J].Applied and Environmental Microbiology,1999,65(8):3754-3756.
[8]Grey B E,Steck T R.The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection[J].Applied and Environmental Microbiology,2001,67(9):3866-3872.
[9]潘乐,孟庆峰,钱爱东,等.VBNC状态细菌检测方法的研究进展[J].中国农学通报,2012,28(5):32-35.
[10]Nocker A,Cheung C Y,Camper A K.Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs dead bacteria by selective removal of DNA from dead cells[J].Journal of Microbiological Methods,2006,67(2):310-320.
[11]Wang Luxin,Mustapha A.EMA-real-time PCR as a reliable method for detection of viable salmonella in chicken and eggs[J].Journal of Food Science,2010,75(3):M134-M139.
[12]熊书,殷幼平,王芳,等.基于EMA-qPCR的茄科青枯菌活体检测技术的建立[J].微生物学通报,2013,40(9):1723-1732.
[13]Ramamurthy T,Ghosh A,Pazhani G P,et al.Current perspectives on viable but nonculturable(VBNC)pathogenic bacteria[J].Frontiers in Public Health,2014,2:103.
[14]Trevors J T.Viable but nonculturable(VBNC)bacteria:Gene expression in planktonic and biofilm cells[J].Journal of Microbiological Methods,2011,86(2):266-273.
[15]Oliver J D.The viable but nonculturable state in bacteria[J].The Journal of Microbiology,2005,43:93-100.
[16]Oliver J D,Bockian R.In vivo resuscitation,and virulence towards mice,of viable but nonculturable cells of Vibrio vulnificus[J].Applied and Environment Microbiology,1995,61(7):2620-2623.
[17]Biosecurity Australia.Revised draft import risk analysis report for the importation of cavendish bananas from the philippines,part C[R].Canberra:Biosecurity Australia,2007.
[18]Zanón M J,Font M I,JordáC.Use of tomato crop residues into soil for control of bacterial wilt caused by Ralstonia solanacearum[J].Crop Protection,2011,30(9):1138-1143.
[19]Paret M L.Management of bacterial wilt of ginger(Zingiber officinale R.)caused by Ralstonia solanacearum with plant essential oils[D].Hawaii:University of Hawaii,2009.
[20]Alfano J R,Collmer A.The typeⅢ(Hrp)secretion pathway of plant pathogenic bacteria:trafficking harpins,Avr proteins,and death[J].Journal of Bacteriology,1997,179(18):5655-5662.