摘要
2009年下半年从一位瑞典病人身上发现一种对几乎所有抗生素耐药的“超级细菌”,该病人因在印度感染肺炎克雷伯菌与大肠杆菌而就医,该细菌对碳青霉烯类(carbapenems)抗生素产生耐药性。超级细菌先在印度发现,后迅速传播至巴基斯坦、孟加拉国、英国、澳大利亚、日本等等,目前已传遍全球。因此超级细菌的预防、检测、治疗是迫在眉睫的事。
在我国,发烧——炎症——给抗生素,已经形成了一种医疗习惯。据调查,中国真正需要使用抗生素的病人不到20%,80%以上属于滥用抗生素。据统计,我国每年有8万人直接、间接死于滥用抗生素。我国7岁以下儿童因为不合理使用抗生素造成耳聋的数量多达30万,占总体聋哑儿童的30%至40%。在住院的感染病患者中,耐药菌感染的病死率为11.7%,普通感染的病死率只有5.4%。这些数字使中国成为世界上滥用抗生素问题最严重的国家之一。超级细菌的出现对我国滥用抗生素的现状再一次敲响了警钟。
对付感染性疾病,除了抗生素之外,我国还有中医。中医治疗感染性疾病的机理肯定与抗生素大不相同,虽然不同在那里还不是很清楚。所以中药也许能成为我们的一颗救星。鉴于此,我们必须尽快涉猎中医药对付超级细菌的研究方向。
在本论文中,我们通过体外合成DNA的方法合成了NDM-1超级细菌的DNA,将NDM-1基因插入PUC57质粒中,然后建立了NDM-1基因的检测方法。再用NDM-1重组质粒转化如大肠杆菌中,用该含有NDM-1重组质粒的大肠杆菌左右筛选具有超级细菌抗性的体外模型对中药黄连进行了评价。
1超级细菌NDM-1基因的体外合成
参照Genbank已公布的NDM-1基因序列(Genbank Accenssion Number:FN396876)进行人工合成,在全长813bp的NDM-1基因的两端分别加上BamHI和EcoRI酶切位点。设计NDM-1全长引物,利用OVERLAP方法形成模版DNA,再利用PCR扩增的方法得到双链DNA,然后将PCR产物转化克隆至克隆载体PUC57中,转化入DH5α大肠杆菌中进行大量扩增。该合成过程交由上海博尚生物技术有限公司完成。后经PCR、酶切和测序鉴定,证实得到了含有准确无误的NDM-1基因的重组质粒。这一质粒当然可用作发展NDM-1基因检测方法的工具。
2 NDM-1基因定量PCR扩增
以前面实验中的NDM-1重组子质粒作为标准品,提取质粒,以紫外分光光度计检测其浓度,再根据其分子量计算摩尔浓度,获得已知拷贝数的NDM-1基因重组质粒标准品。根据NDM-1基因序列设计TaqMan探针和引物并合成,然后以NDM-1基因重组质粒标准品进行10倍梯度稀释,稀释9个浓度分别为:1e10、1e9、1e8、1e7、1e6、1e5、1e4、1e3、1e2copies/ml。批内差异分析者每个浓度梯度12个复孔同时进行定量PCR检测,批间差异分析者分12次,每次每个浓度梯度1孔进行定量PCR;根据定量结果计算批间差异和批内差异。结果显示,这种NDM-1检测方法的检测下限为1e3copies/ml,且重复性良好;其批内差异为0.55%-1.43%之间,其批间差异为0.76%-2.41%之间,其批间差异和批内差异都在允许范围之内。说明本实验所建立的定量PCR检测NDM-1基因的方法敏感性重复性良好。
3 LAMP法检测超级细菌NDM-1基因
根据NDM-1基因序列设计LAMP引物,然后以前面实验获得的NDM-1重组质粒标准品为扩增对象,以1e3的标准品最小浓度进行扩增,并用去离子水作为阴性对照,用LAMP法进行扩增。结果显示,NDM-1重组质粒标准品成功扩增出了LAMP特异性条带,而阴性对照则未见条带,说明我们用LAMP方法成功扩增了NDM-1基因。
4 NDM-1重组质粒的抗生素耐药及中药的影响
将NDM-1基因克隆入具有氨苄青霉素抗性的PUC57质粒中,将该含有NDM-1基因的重组质粒转化入大肠杆菌中,结果该细菌不但具有氨苄青霉素抗性,也具有了卡那霉素抗性,而对照的不含NDM-1质粒的PUC57质粒则只具有氨苄青霉素抗性,而不具有卡那霉素抗性,说明NDM-1基因确实导致了细菌具有抗生素抗性。
接下来我们又进行了中药黄连抑制抗生素抗性细菌的实验,我们用黄连水煎液涂抹在抗生素平板上,然后再将含有NDM-1基因重组质粒工程菌涂抹于平板上,结果显示,涂有黄连的平板上,含有NDM-1基因重组质粒的工程菌只有极少菌落长出,而没有涂黄连的平板上则有大量菌落长出。说明黄连可能抑制含有NDM-1基因重组质粒工程菌的生长。
全文结论
1成功建立了超级细菌NDM-1基因的实时定量PCR检测方法。
2成功建立了超级细菌NDM-1基因的LAMP检测方法。
3含有NDM-1基因的重组质粒转化入大肠杆菌后可使大肠杆菌获得抗生素抗性,可以作为抗超级细菌的药物筛选模型。
4中药黄连具有明显抑制超级细菌NDM-1基因导致的抗生素抗性的作用。
A kind of bacteria was found from a Sweden patient in the second half of 2009 which are resist to almost all of the antibiotics and was named super-bacteria.. This patient was seen doctor for infected by Klebsiella pneumoniae and Bacterium coli. But the bacteria was found to resist to carbapenems. The super-bacteria was firstly occurred in India and then quickly spreaded to Pakistan, Bangladesh, British, Austrilia, Japan and so on. It is spread to all over the world at present. So it is very urgent to research the preservation, testing, therapy of the super-bacteria.
The fever-inflammation-antibiotics prescription was formed to a kind of medical custom in China. According to the investigation, the patients actually need the antibiotic administration are not more than 20% of the total patients. So the population of antibiotic abuse up to 80%. It is reported that there are 80 thousand patients dead for directly or indirectly antibiotic abuse. There are 300 thousand children below 7 years old are suffered from deafness because of antibiotic abuse which account for 30-40% of total deafness children. The mortality of the antibiotic resistant bacteria infected patients account for 11.7% of the total infected patients in hospital, While the mortality of general infected patients only is 5.4%. These data make the china be the world's most serious country of antibiotic abuse. The occurrence of the super-bacteria alarms the China's situation of antibiotic abuse.
Beside the antibiotics, the therapy to Deal with infectious diseases also have Chinese medicine in China. The mechanisms of Chinese Medicine to treat infectious diseases are significantly different from antibiotics. So Chinese Medicine should be the emancipator. We should conduct the research of Chinese medicine to treat the super-bacteria as soon as possible.
In this research, We got the NDM-1 DNA through in vivo synthesis. The NDM-1 DNA was inserted to PUC57 plasmid and then the assays of NDM-1 gene testing were established. The NDM-1 recombinant was transformed into E coli which used to evaluate the anti-super-bacteria activity of Chinese Medicine Coptis chinensis Franch. 1 Super-bacteria NDM-1 DNA synthesis
According to the NDM-1 DNA sequences released in the Genbank, the NDM-1 DNA was synthesized by a commercial corporation. The BamHI and EcoRI enzyme cutting site were added to the 5'and 3'site of the DNA. The primers that cover the whole NDM-1 DNA were designed and template DNA synthesized through overlap method. Then the double strand DNA can get through PCR amplification. The PCR product was purified and cloned to PUC57 plasmid and transformed into DH5a Escherichia coli for quantitatively amplification. The recombinant was verified through PCR, enzyme cutting and sequencing and proved that the NDM-1 gene was correctly inserted into PUC57 plasmid. So this recombinant certainly can be a standard template for developing the NDM-1 gene testing assay.
2Quantitative PCR test of the NDM-1 gene
The NDM-1 recombinant plasmid was extracted from DH5a Escherichia coli. The concentration of the plasmid was tested with ultraviolet spectrophotometer. The NDM-1 gene copy Number was calculated according to the molecular weight of the NDM-1 recombinant. So we got the NDM-1 standard template. The NDM-1 standard template was conducted 10 fold dilution. The standard template was diluted to 9 concentrations as 1e10、1e9、1e8、1e7、1e6、1e5、1e4、1e3、1e2copies/ml. The intrassay was analyzed through test 12 times in every concentration per run. The inter-assay was analyzed through 12 assay runs. The result showed that the lowest concentration of this method can test is 1e3 copies/ml with favourable reproducibility. The intrassay is between 0.55% and 1.43% while the interassay is between 0.76% and 2.41%.Both the intrassay and interassay are within the permissible limit. So we concluded that this quantitative PCR method have a good reproducibility and susceptibility.
3NDM-1 super-bacteria tested with LAMP method
The LAMP primer set was designed according to the NDM-1 DNA sequence. Use the NDM-1 recombinant plasmid as the standard template. The concentration of the standard template is 1e3 copies/ml. Take the deionized water as negative control. The standard template and the negative control were amplified with LAMP method simultaneously. The results showed that there was LAMP special straps in the electropherogram of standard template, while the result of the negative control showed no strap. So we can conclude that we had successfully amplified the NDM-1 gene with LAMP method.
4 NDM-1 gene that resistant to antibiotic and the effect of Chinese Medicine.
We cloned NDM-1 gene to PUC57 plasmid and transformed the recombinant to DH5a Escherichia coli resulted in the DH5a Escherichia coli not only resistant to ampicillin but also resistant to kanamycin, While the PUC57 plasmid not inserted with NDM-1 gene noly resistant to ampicillin. So it is the NDM-1 gene that caused the resistance to kanamycin.
Then we do the experiments about the Chinese Medicine Coptis chinensis Franch inhibit NDM-1 gene caused antibiotic resistance. We smear the Coptis chinensis Franch on the antibiotic LB pad. Then the DH5a Escherichia coli include or not include NDM-1 recombinant plasmids were smeared to the pad. The results showed that there were only a few colony occurred in the pad which was smeared the Coptis chinensis Franch, while the pad have no Coptis chinensis Franch occurred plenty of colony. So the Coptis chinensis Franch can inhibit the growing of DH5a Escherichia coli which include NDM-1 recombinant plasmids.
Conclusion
1 The real time quantitative PCR method to test the NDM-1 gene of super-bacteria was successfully established.
2The LAMP method to test the NDM-1 gene of super-bacteria was successfully established.
3DH5a Escherichia coli which include NDM-1 recombinant plasmids can cause resistant to antibiotic and this Escherichia coli can be a kind of model for super-bacteria inhibition drug screening.
4The Chinese Medicine Coptis chinensis Franch can significantly inhibit the resistance to antibiotic caused by super-bacteria NDM-1 gene.
引文
[1]Petersen AD, Walker RD, Bowman MM, Schott HC 2nd, Rosser EJ Jr. Frequency of isolation and antimicrobial susceptibility patterns of Staphylococcus intermedius and Pseudomonas aeruginosa isolates from canine skin and ear samples over a 6-year period (1992-1997). J Am Anim Hosp Assoc.2002 Sep-Oct;38(5):407-13.
[2]Martinez M, Silley P. Antimicrobial drug resistance. Handb Exp Pharmacol. 2010;(199):227-64.
[3]牟善初.抗生素的回顾、前瞻及其在下呼吸道感染的使用策略.临床误诊误治,1998,11(2)B65
[4]Martinez M, Silley P. Antimicrobial drug resistance. Handb Exp Pharmacol. 2010;(199):227-64.
[5]Livermore DM, Mushtaq S, Warner M, Zhang JC, Mahar jan S, Doumith M, Woodford N. Activity of aminoglycosides, including ACHN-490, against carbapenem-resistant Enterobacteriaceae isolates. J Antimicrob Chemother. 2010 Nov 14. [Epub ahead of print]
[6]Pascoalinho D, Vilas MJ, Coelho L, Moreira P. Linezolid-related immune-mediated severe thrombocytopenia. Int J Antimicrob Agents.2010 Nov 12. [Epub ahead of print]
[7]郑卫.抗细菌药物研究的新进展.国外医药抗生素分册,2000,21(2)B57
[8]Spratt BG. Resistance to antibiotics mediated by target alterations. Science.1994 Apr 15;264(5157):388-93.
[9]Davies J. Inactivation of antibiotics and the dissemination of resistance genes. Science.1994 Apr 15;264(5157):375-82.
[10]Nikaido H. Prevention of drug access to bacterial targets:permeability barriers and active efflux. Science.1994 Apr 15;264(5157):382-8
[11]van Veen HW, Putman M, Margolles A, Sakamoto K, Konings WN. Structure-function analysis of multidrug transporters in Lactococcus lactis. Biochim Biophys Acta.1999 Dec 6;1461(2):201-6.
[12]Rodriguez Noriega E, Morfin Otero R, Esparza Ahumada S. Beta-lactamase production and the patterns of bacterial resistance,1988-1991. Gac Med Mex. 1994 Sep-Oct;130(5):355-60.
[13]Huletsky A, Couture F, Levesque RC. Nucleotide sequence and phylogeny of SHV-2 beta-lactamase. Antimicrob Agents Chemother.1990 Sep;34(9):1725-32.
[14]Bush K. Characterization of beta-lactamases. Antimicrob Agents Chemother. 1989 Mar;33(3):259-63.
[15]Bush K. Classification of beta-lactamases:groups 1,2a,2b, and 2b' Antimicrob Agents Chemother.1989 Mar;33(3):264-70.
[16]Bush K. Classification of beta-lactamases:groups 2c,2d,2e,3, and 4. Antimicrob Agents Chemother.1989 Mar;33(3):271-6.
[17]Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother.1995 Jun;39(6):1211-33.
[18]Charpentier E, Tuomanen E. Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae. Microbes Infect.2000 Dec;2(15):1855-64.
[19]Reynolds PE. Structure, biochemistry and mechanism of action of glycopeptide antibiotics. Eur J Clin Microbiol Infect Dis.1989 Nov;8 (11):943-50.
[20]Arthur M, Reynolds P, Courvalin P. Glycopeptide resistance in enterococci.Trends Microbiol.1996 Oct;4(10):401-7.
[21]Leclercq R, Dutka-Malen S, Duval J, Courvalin P. Vancomycin resistance gene vanC is specific to Enterococcus gallinarum. Antimicrob Agents Chemother. 1992 Sep;36(9):2005-8.
[22]Navarro F, Courvalin P. Analysis of genes encoding D-alanine-D-alanine ligase-related enzymes in Enterococcus casseliflavus and Enterococcus flavescens. Antimicrob Agents Chemother.1994 Aug;38(8):1788-93.
[23]Arthur M, Reynolds PE, Depardieu F, Evers S, Dutka-Malen S, Quintiliani R Jr, Courvalin P. Mechanisms of glycopeptide resistance in enterococci. J Infect.1996 Jan;32(1):11-6.
[24]Arthur M, Quintiliani R Jr. Regulation of VanA- and VanB-type glycopeptide resistance in enterococci. Antimicrob Agents Chemother.2001 Feb;45(2):375-81.
[25]Clark NC, Teixeira LM, Facklam RR, Tenover FC. Detection and differentiation of vanC-1, vanC-2, and vanC-3 glycopeptide resistance genes in enterococci. J Clin Microbiol.1998 Aug;36(8):2294-7.
[26]Walsh C. Molecular mechanisms that confer antibacterial drug resistance. Nature.2000 Aug 17;406(6797):775-81.
[27]Gholizadeh Y, Courvalin P. Acquired and intrinsic glycopeptide resistance in enterococci. Int J Antimicrob Agents.2000 Nov;16 Suppl 1:S11-7.
[28]Billot-Klein D, Gutmann L, Sable S, Guittet E, van Heijenoort J. Modification of peptidoglycan precursors is a common feature of the low-level vancomycin-resistant VANB-type Enterococcus D366 and of the naturally glycopeptide-resistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides, and Enterococcus gallinarum. J Bacteriol.1994 Apr;176(8):2398-405.
[29]Leclercq R, Courvalin P. Bacterial resistance to macrolide, lincosamide, and streptogramin antibiotics by target modification. Antimicrob Agents Chemother.1991 Jul;35(7):1267-72.
[30]Roberts MC, Sutcliffe J, Courvalin P, Jensen LB, Rood J, Seppala H. Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants. Antimicrob Agents Chemother.1999 Dec;43(12):2823-30.
[31]Ross JI, Eady EA, Cove JH, Cunliffe WJ, Baumberg S, Wootton JC. Inducible erythromycin resistance in staphylococci is encoded by a member of the ATP-binding transport super-gene family. Mol Microbiol.1990 Jul;4(7):1207-14.
[32]Vester B, Douthwaite S. Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrob Agents Chemother.2001 Jan;45(1):1-12.
[33]Vaara M. Agents that increase the permeability of the outer membrane. Microbiol Rev.1992 Sep;56(3):395-411.
[34]Mingeot-Leclercq MP, Glupczynski Y, Tulkens PM. Aminoglycosides: activity and resistance. Antimicrob Agents Chemother.1999 Apr;43(4):727-37.
[35]Karlowsky JA, Hoban DJ, Zelenitsky SA, Zhanel GG. Altered denA and anr gene expression in aminoglycoside adaptive resistance in Pseudomonas aeruginosa. J Antimicrob Chemother.1997 Sep;40(3):371-6.
[36]Shaw KJ, Rather PN, Hare RS, Miller GH. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev.1993 Mar;57(1):138-63.
[37]Drlica K. Mechanism of fluoroquinolone action. Curr Opin Microbiol.1999 Oct;2(5):504-8.
[38]Borges-Walmsley MI, Walmsley AR. The structure and function of drug pumps. Trends Microbiol.2001 Feb;9(2):71-9.
[39]Bower CK, Daeschel MA. Resistance responses of microorganisms in food environments. Int J Food Microbiol.1999 Sep 15;50(1-2):33-44.
[40]杨继章,杨树民.碳青霉烯类抗生素的开发与临床应用.上海医药,2004,25(3):109-112.
[41]林彩,刘松青.美罗培南的药理及临床应用.中国药业,2003,12(8):72-74.
[42]崔银珠.新型抗菌药物比阿培南(biapenem).世界临床药物,2003,24(9):572-575.
[43]曹国颖,傅得兴,孙春华.新型碳青酶烯类抗生素 艾他培南.中国临床药理学杂志,2005,21(1):74-76.
[44]梅开城,王睿.碳青霉烯类药物抗生素后效应研究概况.中国新药杂志,1999,8(11):727-730.
[45]Bowker KE, Hols HA, Rccves DS. Bactericidal activity, post antibiotic effect and modified controlled effective regrowth time of meropenem at high concentrations.J Antimicrob Chemother,1996,38(6):1055-1057.
[46]何菊英,刘松青,藏雷.新型碳青霉烯帕尼培南/倍他米隆的药理研究和临床应用.中国新药与临床杂志,2003,22(2):103-106.
[47]王永赓,钱广鸿.碳青霉烯类抗生素的比较及临床应用.基层医学论坛,2007,11(3):265-268.
[48]黄祥,梅巍,邸晓辉.碳青霉烯类抗生素的药动学药效学与临床应用.中国医院药学杂志,2001,21(12):75-78.
[49]扬帆,张婴元,郑丽叶,等.帕尼培南倍他米隆的临床评价.中华内科杂志,2000,39(2):107-110.
[50]张幸国,黄明珠,魏泽庆,等.三种碳青霉烯类抗生素对ICU分离菌株的体外杭菌作用.中国抗生素杂志,2001,26(4):283-286
[51]游学甫,陈慧贞.美罗培南的药理及临床应用.国外医药抗生素分册,2000,21(1):27-29.
[52]Norrby SR, Newell PA, Faulkner KL,et al. Safety profile of meropenem: international clinical experience based on the first 3125 patients treated with meropenem. J Antimicrob Chemother,1995,36(Suppl A):207
[53]Krueger WA, Bulitta J, Kinzig Schippers M, et al. Evaluation by Monte Carlo simulation of the pharmacokinetics of two doses of meropenem administered intermittently or as a continuous infusion in healthy volunteers. Antimicrob Agents Chemother,2005,49(5):1881
[54]Kasiakou SK, Lawrence KR, Choulis N, et al. Continuous versus intermittent intravenous administration of antibacterials with time dependent action:a systematic review of pharmacokinetics and pharmacodynamic parameters. Drugs, 2005,5 (17):2499
[55]Mouton JW, Touzw DJ, Horrevorts AM, et al. Comparative pharmacokinetics of the carbapenems:clinical implications. Clin Pharmacokinet,2000,39 (3):185
[56]张石革,藏靖,梁建华,等.碳青霉烯类抗生素的研究进展及其在抗感染中的应用评价.中国医院用药评价与分析,2004,4(2):71-73.
[57]Limanaky AS,Musi MA, Viale AM.Loss of a 29 kilodalton outer membrane protein in Acinetobacter baumarmii is associated with imipenecn resistance.J Clin Microbiol,2002,40(12):4776
[58]Nordmarm P,Poirel L. Emerging carbapenemsase in Gram negative aerobes.Clin Microbiol Infect,2002,8(6):321
[59]李向平.执业药师在合理使用抗菌药物中的地位和作用.中国当代医学,2004,3(6):45-48.
[60]范玉峰.不合理应用抗生素的现状与对策.中国医药导报.2006。10(3):30-33.
[61]杨晓华.1230例抗生素不良反应分析.中国药房2001,12(2):106-107.
[62]胡锦芳,黄春梅,王劲柏.临床抗菌药物不合理应用的调查分析.实用临床医学.2004.5(6):41-44.
[63]宋丹妮,谢惠嫒.对当前抗生素滥用现状的一些看法.广东微量元素科学,2005,13(2):14-16.
[64]梁奕宏,柳红.抗生素滥用——导致疾病和死亡的社会问题.中国检验检疫,2004.(10):59-60.
[65]邱家学.浅析我国抗生素滥用现状及危害.上海医药.2006,27(1):22-25.
[66]赵红梅,金升藻.滥用抗生素对人畜的危害及对策研究.国外医药抗生素分册,2003.24(4):164-167
[67]王来成.防止滥用抗生素.江苏药学与临床研究,2003,11(4):40-41.
[68]宋艳,贾春风.关于抗生素滥用问题及对策[J].中国现代医药杂志.2006,8(11):3-5.
[69]赵民生,一王会贞.滥用抗生素的危害与根源[J].食品与药品.2006,8(11):34-38.
[70]李丽萍.抗生素滥用的原因分析.山西职工医学院学报。2005,15(2):15-17.
[71]Spratt BG. Resistance to antibiotics mediated by target alterations. Science.1994 Apr 15;264(5157):388-93.
[72]Dancer SJ, Shears P, Platt DJ. Isolation and characterization of coliforms from glacial ice and water in Canada's High Arctic. J Appl Microbiol.1997 May;82(5):597-609.
[73]Ferber D. New hunt for the roots of resistance. Science.1998 Apr 3;280(5360):27.
[74]Mingeot-Leclercq MP, Glupczynski Y, Tulkens PM. Aminoglycosides: activity and resistance. Antimicrob Agents Chemother.1999 Apr;43(4):727-37.
[75]Levy SB. Active efflux mechanisms for antimicrobial resistance. Antimicrob Agents Chemother.1992 Apr;36(4):695-703.
[76]Borges-Walmsley MI, Walmsley AR. The structure and function of drug pumps. Trends Microbiol.2001 Feb;9(2):71-9.
[77]Farrow KA, Lyras D, Rood JI. The macrolide-lincosamide-streptogramin B resistance determinant from Clostridium difficile 630 contains two erm(B) genes. Antimicrob Agents Chemother.2000 Feb;44(2):411-413.
[78]高美英.2003.细菌对抗生素耐药性的起源分子基础与对策.医药导报,22(1):6~8
[79]傅文栋.中草药及其有效成分体外抗菌抗病毒研究进展[J].中国兽医药杂志,2006,5:66-68.
[80]李建志.黑龙江省地产中药抗菌作用实验研究[J].中医药学报,2005,33(6):24-27.
[81]李仲兴,王秀华,张明明,等.五倍子乙醇提取物对140株肠球菌体外抗菌活性观察[J].中国中医药信息杂志,2004,11(3):201-204.
[82]周峰,籍保平,李博,等.十二种中药挥发油及其滤液体外抗菌活性研究[J]食品科学,2005,3:50-53.
[83]YakubuM Bello, Odama LE, N andita B De. Studies on the Antibacterial Activity of the Extract of Stachytarpheta angustifolia. Journal of Nanjing Medical university.2003,17(3):116-119.
[84]Eleyinmi AF. Chemical composition and antibacterial activity of Gongronema latifolium. J Zhejiang Univ Sci B.2007 May;8(5):352-358.
[85]李忠海,钟海雁,郑锦星,等.槟榔提取物在小白鼠体内的抑菌作用[J].食品与机械,2007,23(5):81-84.
[86]蒋锦琴,黄卫平,张小贤,等.中药结核丸对结核分枝杆菌内外抗菌效果的观 察[J].江西医学检验,2005,23(6):521-523.
[87]Ibrahim M, Khan AA, Tiwari SK, Habeeb MA, Khaja MN, Habibullah CM. Antimicrobial activity of Sapindus mukorossi and Rheum emodi extracts against H pylori:In vitro and in vivo studies. World J Gastroenterol.2006 Nov 28;12(44):7136-7142.
[88]Kwon HA, Kwon YJ, Kwon DY, Lee JH. Evaluation of antibacterial effects of a combination of Coptidis Rhizoma, Mume Fructus, and Schizandrae Fructus against Salmonella. Int J Food Microbiol.2008 Sep 30;127(1-2):180-183.
[89]P. K. Tarus, P. H. C oom bes, N. R. Crouch, act. B enzo phenanthridine alkaloids from stem bark of the Forest Knobwood, Zanth oxylumdavyi (Rutaceae)[J]. South African Journ al of Botany.2006,72:555-559.
[90]常银霞,陈业高.蝉翼藤属药用植物的研究进展[J].山西师范大学学报(自然科学版),2005,19(1):95-97.
[91]杨扬,朱顺英,唐李斐,等.羽裂蟹甲草挥发油的化学成分分析及抗菌活性研究[J].武汉大学学报(理学版),2007,53(2):198-200.
[92]梁英,韩鲁佳.黄芩中黄酮类化合物药理学作用研究进展[J].中国农业大学学报,2003,8(6):9-11.
[93]陈秋东,徐蓉,徐志南,等.决明子中蒽醌类化学成分及其生物活性研究进展[J].中国现代应用药学杂志,2003,20(2):120-123.
[94]Gui You CUI, Jun Yan LIU, Ren Xiang TAN. A New Antimicrobial Flavonol Glycoside from Alchornea davidii. Chinese Chemical Letters.2003,14(2): 179-182.
[95]Magina MD, Dalmarco EM, Wisniewski A Jr, Simionatto EL, Dalmarco JB, Pizzolatti MG, Brighente IM. Chemical composition and antibacterial activity of essential oils of Eugenia species. J Nat Med.2009 Jul;63(3):345-350.
[96]严梅桢,左风,宋红月,等.黄芩汤及其代谢产物抗菌作用的比较研究[J].中国中药杂志,2003,28(3):243-245.
[97]覃筱燕,张淑萍,杨林,等.复方双花口服液的解热、抗炎、抑菌作用的实验研究[J].中央民族大学学报(自然科学版),2003,12(4):315-318.
[98]卢芳国,朱应武,田道法,等.12个中药复方体外抗菌作用的研究[J].湖南中医学院学报,2004,24(4):9-12.
[99]毛理纳,罗予,胡新辉,等.黄连和大黄联合头孢他啶体内外抗菌作用[J]. 中药药理与临床,2006,22(2):38-40.
[100]熊南燕,王雪铃,曹明耀,等.鱼腥草注射液对硫酸庆大酶素兔体内抗菌作用的影响[J].现代中西医结合杂志,2007,24(16):3471-3474.
[101]李文杰,李慧.中药注射剂与某些抗菌药物不宜配伍应用[J].中国药业,2006,15(9):23-26.
[102]王静,张淑文.中药逆转细菌耐药的研究进展[J].临床和实验医学杂志,2007,6(1):153-156.
[103]芦亚君,程宁.3种中药方剂逆转大肠杆菌耐药性的实验观察[J].西北药学杂志,2007,22(6):309-311.
[104]蒋培余.中药抑制剂逆转细菌耐药性的研究进展[J].中国医药学报,2008,10(10):53-55.
[105]Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, Walsh TR. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother.2009 Dec;53(12):5046-5054.
[106]Moellering RC Jr. NDM-1--a cause for worldwide concern. N Engl J Med. 2010 Dec 16:363(25):2377-2379.
[107]Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S, Mushtaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W, Livermore DM, Woodford N. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK:a molecular, biological, and epidemiological study. Lancet Infect Dis.2010 Sep;10(9):597-602.
[108]Rolain JM, Parola P, Cornaglia G. New Delhi metallo-beta-lactamase (NDM-1):towards a new pandemia? Clin Microbiol Infect.2010 Dec;16(12):1699-1701.
[109]牛丹丹,王正祥.全基因合成方法学研究.应用与环境生物学报,2007,13(4):515-518.
[110]]Clegg RM. Fluorescence resonance energy transfer. Curt Opin Biotechnol, 1995,6(1):103-110.
[111]Mikael 1, Jose MA, Martin B, et aL The real-time poly-merase ehain reaction. Molecuiar Aspects of Medicine,2006,27:95-125
[112]曾冰冰,肖凯军,石磊,肖如武LAMP方法在食品微生物检测中的应用.现代食品与药品杂志.2007,17(1):22-25.
[113]刘永生,定耀忠,张杰.环介导等温扩增(LAMP)技术的应用研究.中国农学通报,2010,26(8):87-89.
[114]Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res.2000 Jun 15;28(12):E63.
[115]Parida M, Sannarangaiah S, Dash PK, Rao PV, Morita K. Loop mediated isothermal amplification (LAMP):a new generation of innovative gene amplification technique;perspectives in clinical diagnosis of infectious diseases. Rev Med Virol.2008 Nov-Dec;18(6):407-421.
[116]Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP):a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother.2009 Apr;15(2):62-69.
[117]Chou PH, Lin YC, Teng PH, Chen CL, Lee PY. Real-Time Target-Specific Detection of Loop-Mediated Isothermal Amplification for White Spot Syndrome Virus Using Fluorescence Energy Transfer-Based Probes. J Virol Methods.2011 Jan 20. [Epub ahead of print]
[118]Nakauchi M, Yoshikawa T, Nakai H, Sugata K, Yoshikawa A, Asano Y, Ihira M, Tashiro M, Kageyama T. Evaluation of reverse transcription loop-mediated isothermal amplification assays for rapid diagnosis of pandemic influenza A/H1N12009 virus. J Med Virol.2011 Jan;83(1):10-15.
[119]Fang X, Chen H, Yu S, Jiang X, Kong J. Predicting Viruses Accurately by a Multiplex Microf luidic Loop-Mediated Isothermal Amplification Chip. Anal Chem.2010 Dec 10. [Epub ahead of print]
[120]Poirel L, Lagrutta E, Taylor P, Pham J, Nordmann P. Emergence of metallo-ss-lactamase NDM-1-producing multidrug resistant Escherichia coli in Australia. Antimicrob Agents Chemother.2010 Nov;54(11):4914-6.