幽门螺杆菌菌落PCR的优化
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摘要
目的:通过对常规菌落PCR方法进行优化,探索提高幽门螺杆菌(H.pylori)菌落PCR扩增效率的方法。方法:提取H.pylori 26695基因组DNA作为阳性对照组,以未经任何处理的H.pylori 26695菌落作为阴性对照组,经煮沸裂解速冻法获得的H.pylori 26695菌落作为试验组,随机选择8个不同的基因(16S r DNA、cag A-U、cag A-D、ice A、ure A、het A、rop N、Hp0792)作为菌落PCR的候选基因进行菌落PCR验证,最后以1%琼脂糖凝胶进行电泳检测。结果:在选择的目标基因中,阴性对照组未扩增出目的条带,试验组与阳性对照组均扩增出目的相应大小的条带;与常规菌落PCR比较,煮沸速冻裂解法能显著提高H.pylori菌落PCR的扩增效率。结论:煮沸速冻裂解法可用于H.pylori的高通量筛选鉴定和分子诊断。
Objective:To improve the PCR amplification efficiency of Helicobacter pylori(H.pylori colonies by optimizing the conventional colony PCR method.Methods:The genomic DNA from H.pylori 26695 strain was extracted as a positive control group,and H.pylori 26695 colonies without any treatment were used as a negative control group.Lysates derived from boiling-freezing H.pylori26695 colonies were used as experimental groups.Eight genes(16 S rDNA,cagA-U,cagA-D,iceA,ureA,hetA,ropN,Hp0792) were randomly selected for colony PCR.PCR products were finally detected by electrophoresis on a 1% agarose gel.Results:Electrophoresis results show that all selected genes could be amplified in positive control and experimental group,however,it could not be amplified in negative control group.Compared to positive control group,all eight selected genes were also wellamplified in experiment group.Conclusion:The boiling-freezing method can be used for high-throughput screening and molecular diagnosis of H.pylori.
Objective:To improve the PCR amplification efficiency of Helicobacter pylori(H.pylori colonies by optimizing the conventional colony PCR method.Methods:The genomic DNA from H.pylori 26695 strain was extracted as a positive control group,and H.pylori 26695 colonies without any treatment were used as a negative control group.Lysates derived from boiling-freezing H.pylori26695 colonies were used as experimental groups.Eight genes(16 S rDNA,cagA-U,cagA-D,iceA,ureA,hetA,ropN,Hp0792) were randomly selected for colony PCR.PCR products were finally detected by electrophoresis on a 1% agarose gel.Results:Electrophoresis results show that all selected genes could be amplified in positive control and experimental group,however,it could not be amplified in negative control group.Compared to positive control group,all eight selected genes were also wellamplified in experiment group.Conclusion:The boiling-freezing method can be used for high-throughput screening and molecular diagnosis of H.pylori.
引文
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[2]SGAMBATO D,MIRANDA A,ROMANO L,et al. Gut microbiota and gastric disease[J]. Minerva Gastroenterol Dietol,2017,63(4):345-354.
[3] MAKOLA D,PEURA D A,CROWE S E. Helicobacter pylori infection and related gastrointestinal diseases[J]. J Clin Gastroenterol,2007,41(6):548-558.
[4]GENG X,LI W,CHEN Z,et al. The bifunctional enzyme Spo T is involved in the clarithromycin tolerance of Helicobacter pylori by upregulating the transporters HP0939,HP1017,HP0497,and HP0471[J]. Antimicrob Agents Chemother,2017,61(5).
[5]LAI C,FISCHER T,MUNROE E. Homologous recombination using bacterial artificial chromosomes[J]. Cold Spring Harb Protoc,2015,2015(2):180-190.
[6] THUNG I,ARAMIN H,VAVINSKAYA V,et al. Review article:the global emergence of Helicobacter pylori antibiotic resistance[J]. Aliment Pharmacol Ther,2016,43(4):514-533.
[7]PACKEISER H,LIM C,BALAGURUNATHAN B,et al.An extremely simple and effective colony PCR procedure for bacteria,yeasts,and microalgae[J]. Appl Biochem Biotechnol,2013,169(2):695-700.
[8]YAN M,LI W,ZHOU Z,et al. Direct detection of various pathogens by loop-mediated isothermal amplification assays on bacterial culture and bacterial colony[J]. Microb Pathog,2017,102:1-7.
[9]WEISS D,ENGELMANN I,BRAUN S D,et al. A multiplex real-time PCR for the direct,fast,economic and simultaneous detection of the carbapenemase genes bla KPC,bla NDM,bla VIM and bla OXA-48[J]. J Microbiol Methods,2017,142:20-26.
[10]CUI G Z,ZHANG J,HONG W,et al. Improvement of Clos Tron for successive gene disruption in Clostridium cellulolyticum using a pyr F-based screening system[J].Appl Microbiol Biotechnol,2014,98(1):313-323.
[11]SHAHMORADI M,FARIDIFAR P,SHAPOURI R,et al. Determining the biofilm forming gene profile of Staphylococcus aureus clinical isolates via multiplex colony PCR method[J]. Rep Biochem Mol Biol,2019,7(2):181-188.
[12]JAMAL M,SHARMA S P,CHUNG H J,et al. Ultrahigh efficient colony PCR for high throughput screening of bacterial genes[J]. Indian J Microbiol,2017,57(3):365-369.
[13]WAN M,ROSENBERG J N,FARUG J,et al. An improved colony PCR procedure for genetic screening of chlorella and related microalgae[J]. Biotechnol Lett,2011,33(8):1615-1619.
[14]MOHR G,HONG W,ZHANG J,et al. A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum[J]. PLo S One,2013,8(7):e69032.
[15]AZHAHIANAMBI P,GHOSH S,ASHOK K C,et al.Cost effectiveness of colony lysis and colony PCR methods for screening of recombinant Escherichia coli colonies a comparative study[J]. Indian J Exp Biol,2008,46(10):731-735.
[16]LIN X,YANG F,ZHOU Y,et al. Highly-efficient colony PCR method for red yeasts and its application to identify mutations within two leucine auxotroph mutants[J].Yeast,2012,29(11):467-474.
[17]WALCH G,KNAPP M,RAINER G,et al. Colony-PCR is a rapid method for DNA amplification of hyphomycetes[J]. J Fungi(Basel),2016,2(2):212-215.
[18]GUO C,LIU F,ZHU L,et al. Analysis of culturable microbiota present in the stomach of children with gastric symptoms[J]. Braz J Microbiol,2019,50(1):107-115.
[19]CHEN S Y,ZHANG R G,DUAN G C. Pathogenic mechanisms of the oncoprotein Cag A in H. pylori-induced gastric cancer(Review)[J]. Oncol Rep,2016,36(6):3087-3094.
[20]MA Y J,DUAN G C,ZHANG R G,et al. Mutation of ice A in Helicobacter pylori compromised IL-8 induction from human gastric epithelial cells[J]. J Basic Microbiol,2010,50(Suppl 1):83-88.
[21]PEEK R J,THOMPSON S A,DONAHUE J P,et al.Adherence to gastric epithelial cells induces expression of a Helicobacter pylori gene,ice A,that is associated with clinical outcome[J]. Proc Assoc Am Physicians,1998,110(6):531-544.
[22]ZHAO H,WU Y,XU Z,et al. Mechanistic insight into the interaction between Helicobacter pylori urease subunit alpha and its molecular chaperone Hsp60[J]. Front Microbiol,2019,10:153.
[23]HU L T,FOXALL P A,RUSSELL R,et al. Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ure A and ure B[J]. Infect Immun,1992,60(7):2657-2666.
[24]TSANG J,HOOVER T R. Basal body structures differentially affect transcription of Rpo N-and Fli A-dependent flagellar genes in Helicobacter pylori[J]. J Bacteriol,2015,197(11):1921-1930.
[25]SAHIN F,KIYAN M,KARASARTOVA D,et al. A new method for the disruption of cell walls of gram-positive bacteria and mycobacteria on the point of nucleic acid extraction:sand method[J]. Mikrobiyol Bul,2016,50(1):34-43.
[26]ALSHAHNI M M,MAKIMURA K,YAMADA T,et al.Direct colony PCR of several medically important fungi using ampdirect plus[J]. Jpn J Infect Dis,2009,62(2):164-167.
[27]WANG F,MENG W,WANG B,et al. Helicobacter pylori-induced gastric inflammation and gastric cancer[J].Cancer Lett,2014,345(2):196-202.
[28]BUGAYTSOVA J A,BJORNHAM O,CHERNOV Y A,et al. Helicobacter pylori adapts to chronic infection and gastric disease via p H-responsive Bab A-mediated adherence[J]. Cell Host Microbe,2017,21(3):376-389.