超级细菌NDM-1基因检测方法、抑菌评价模型的建立及黄连抑菌探索试验的研究
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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.
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
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