非培养方法结合ERIC-PCR研究肝硬化腹水患者肠道真菌菌群多样性
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摘要
【目的】
多年来,在医学微生态学研究领域中,人们在肠道真菌菌群构成的多样性上,鲜有深入研究。然而在临床上,胃肠道菌群失衡引起肠道真菌感染的发病率、死亡率却不断增高。目前,大多数实验室建立的鉴定方法是将临床粪便标本在培养阳性的基础上再进行研究,但有些真菌并不能在培养基上生长,使得培养阴性的含菌标本不能被用来鉴定菌种。这就限制了该方法的应用范围。因此,从粪便标本中直接提取真菌总DNA进行研究更能全面真实反映肠道真菌微生物区系组成结构。本研究选用提取效果得到较多肯定的QIAamp~(?)DNAStool Mini Kit结合玻璃珠击打法(QIA+玻珠法)来抽提粪便中的总DNA,然后用真菌通用引物18S rDNA PCR对真菌进行检测,同时应用克隆测序方法分析上述方法获得肠道真菌DNA的抽提效率及质量,并将其与实验室常规真菌分离培养鉴定的结果进行比较。在此基础上,用克隆测序建立系统发育树及ERIC-PCR指纹图技术对肝硬化腹水患者粪便标本中肠道真菌基因组DNA的多样性进行研究。
【材料与方法】
1.收集28例肝硬化腹水患者、20例慢性乙肝非活动期患者、30例健康人的尾便,一份用实验室常规真菌分离培养鉴定法鉴定到种,另一份1小时内保存于-80℃。
2.用QIA+玻珠法抽提保存于-80℃的粪便的总DNA,然后用真菌通用引物18S rDNA PCR方法扩得真菌总DNA,随机挑取8份来自肝硬化腹水患者的23个克隆子测序建立系统发育树,并与实验室常规菌种鉴定结果比对。
3.用ERIC-PCR指纹图技术分析比较9例肝硬化腹水患者、8例慢乙肝非活动期患者、8例健康人的肠道真菌基因组DNA的多样性。
【结果】
1.相比于真菌分离培养鉴定法,真菌通用引物18S rDNA PCR技术结合QIA+玻珠法(通用引物PCR检测法)可以快速、敏感地检测到粪便标本中的真菌定植或感染。同时无论是通用引物PCR检测法还是真菌分离培养鉴定法,肝硬化腹水患者粪便真菌的检出率都高于慢乙肝非活动期患者和健康人。
2.8例肝硬化腹水患者的标本,真菌分离培养鉴定法只鉴定出3种念珠菌,而通用引物PCR检测法从中随机挑选23个克隆子通过建立系统发育树分析却发现了5种念珠菌、1种曲霉、1种酿酒酵母和1个被孢霉属。
3.ERIC-PCR指纹图技术分析提示肝硬化腹水患者肠道真菌菌群多样性较丰富,而健康人和慢乙肝非活动期患者的肠道真菌菌群数目较少。
【结论】
1.通用引物PCR检测法可以从粪便中获取高质量的真菌总DNA,有利于肠道真菌菌群构成的多样性研究。
2.肝硬化腹水患者肠道真菌菌群的多样性比较丰富,念珠菌为优势菌。
【Objective】
In the past years,in the field of microecology,people have did little deep work to the fundemental problems such as diversification and ecological function of gastrointestinal fungus flora composition,however,there is a constantly increase of incidence and mortality rate due to the imbalance of gastrointestinal bacterial communities(flora)in clinic in recent years.At present,most of the laboratory around the world do their research based on the positive result of excremental culture,but we have to admit that some of the fungus can not grow on any culture medium,and thus the negative culture result can not be used to characterize strain it belongs to,so this defect restrain its application scope.For the reason mentioned above,directly extract total fungal DNA from feces sample could be a alternative strategy to comprehensively reflect the intestinal microflora.in this study we choose the QIAamp~(?)DNA Stool Mini Kit combined with bead-beating,a widely confirmed method for extracting the total DNA from feces,and then we use universal primer 18S rDNA PCR to detect fungi DNA,meanwhile,random cloning and sequence analysis was also demonstrated the quality of fungal DNA extracts using the above method.And the result of this method is also compared with the fungal isolation and culture identification methods Futher more,on the basis of the above, establishment of phylogenetic trees by cloning and sequencing and ERIC-PCR fingerprint map were utilized to analyze the polymorphism of fungal community in ascites of patients with cirrhosis.
【material and method】
1.Collecting stool specimens of 28 Cirrhotic Patients with Ascites、20 Chronic hepatitis B patients(non-active)、30 healthy subjects.One portion was identified to genus by the fungal isolation and culture identification method,the other portion stored in-80℃within 1 hour.
2.Choosing the QIAamp~(?)DNA Stool Mini Kit combined with bead-beating,a widely confirmed method for extracting the total DNA from feces stored in 80℃,and then we use universal primer 18S rDNA PCR to detect fungi DNA,Randomly selected 23 clones from 8 ascites of patients with cirrhosis to establish of phylogenetic trees,and the result of this method is also compared with the fungal isolation and culture identification method.
3.Using ERIC-PCR fingerprint technique to analysis and compare the diversity of fungal flora in the 9 ascites of patients with cirrhosis、 8 Chronic hepatitis B patients(non-active)and 8 healthy subjects.
【Result】
1.Compaired with the fungal isolation and culture identification method,universal primer 18S rDNA PCR combined with QIA+bead beating(universal primer PCR detection method)is more fastly and sensitively to detect fungal colonization and infection of digestive tract,meanwhile,both of them showed that the detective rate of stool fungi from Ascites of Patients with Cirrhosis is higher than Chronic hepatitis B patients(non-active)and healthy subjects.
2.In 8 ascites of patients with cirrhosis's stool specimens,there were 3 candida spp.found by common fungi culture method.but by establishing phylogenetic trees,there were found 5 candida spp.、one Aspergiilus spp.、one Saccharomyces cerevisiae and one Mortierella spp.
3.ERIC-PCR fingerprint technique suggested that:the polymorphism of gastrointestinal fungal community in ascites of Patients with Cirrhosis was more abundant,compared with the Chronic hepatitis B patients(non-active)and healthy subjects.
【Conclusions】
1.Universal primer PCR detection method could extract high quality fungal total DNA from feces,which is beneficial to study on the diversification of gastrointestinal fungus flora composition.
2.the polymorphism of gastrointestinal fungal community in ascites of Patients with Cirrhosis was relatively rich,and Candida spp.is the dominant fungi.
多年来,在医学微生态学研究领域中,人们在肠道真菌菌群构成的多样性上,鲜有深入研究。然而在临床上,胃肠道菌群失衡引起肠道真菌感染的发病率、死亡率却不断增高。目前,大多数实验室建立的鉴定方法是将临床粪便标本在培养阳性的基础上再进行研究,但有些真菌并不能在培养基上生长,使得培养阴性的含菌标本不能被用来鉴定菌种。这就限制了该方法的应用范围。因此,从粪便标本中直接提取真菌总DNA进行研究更能全面真实反映肠道真菌微生物区系组成结构。本研究选用提取效果得到较多肯定的QIAamp~(?)DNAStool Mini Kit结合玻璃珠击打法(QIA+玻珠法)来抽提粪便中的总DNA,然后用真菌通用引物18S rDNA PCR对真菌进行检测,同时应用克隆测序方法分析上述方法获得肠道真菌DNA的抽提效率及质量,并将其与实验室常规真菌分离培养鉴定的结果进行比较。在此基础上,用克隆测序建立系统发育树及ERIC-PCR指纹图技术对肝硬化腹水患者粪便标本中肠道真菌基因组DNA的多样性进行研究。
【材料与方法】
1.收集28例肝硬化腹水患者、20例慢性乙肝非活动期患者、30例健康人的尾便,一份用实验室常规真菌分离培养鉴定法鉴定到种,另一份1小时内保存于-80℃。
2.用QIA+玻珠法抽提保存于-80℃的粪便的总DNA,然后用真菌通用引物18S rDNA PCR方法扩得真菌总DNA,随机挑取8份来自肝硬化腹水患者的23个克隆子测序建立系统发育树,并与实验室常规菌种鉴定结果比对。
3.用ERIC-PCR指纹图技术分析比较9例肝硬化腹水患者、8例慢乙肝非活动期患者、8例健康人的肠道真菌基因组DNA的多样性。
【结果】
1.相比于真菌分离培养鉴定法,真菌通用引物18S rDNA PCR技术结合QIA+玻珠法(通用引物PCR检测法)可以快速、敏感地检测到粪便标本中的真菌定植或感染。同时无论是通用引物PCR检测法还是真菌分离培养鉴定法,肝硬化腹水患者粪便真菌的检出率都高于慢乙肝非活动期患者和健康人。
2.8例肝硬化腹水患者的标本,真菌分离培养鉴定法只鉴定出3种念珠菌,而通用引物PCR检测法从中随机挑选23个克隆子通过建立系统发育树分析却发现了5种念珠菌、1种曲霉、1种酿酒酵母和1个被孢霉属。
3.ERIC-PCR指纹图技术分析提示肝硬化腹水患者肠道真菌菌群多样性较丰富,而健康人和慢乙肝非活动期患者的肠道真菌菌群数目较少。
【结论】
1.通用引物PCR检测法可以从粪便中获取高质量的真菌总DNA,有利于肠道真菌菌群构成的多样性研究。
2.肝硬化腹水患者肠道真菌菌群的多样性比较丰富,念珠菌为优势菌。
【Objective】
In the past years,in the field of microecology,people have did little deep work to the fundemental problems such as diversification and ecological function of gastrointestinal fungus flora composition,however,there is a constantly increase of incidence and mortality rate due to the imbalance of gastrointestinal bacterial communities(flora)in clinic in recent years.At present,most of the laboratory around the world do their research based on the positive result of excremental culture,but we have to admit that some of the fungus can not grow on any culture medium,and thus the negative culture result can not be used to characterize strain it belongs to,so this defect restrain its application scope.For the reason mentioned above,directly extract total fungal DNA from feces sample could be a alternative strategy to comprehensively reflect the intestinal microflora.in this study we choose the QIAamp~(?)DNA Stool Mini Kit combined with bead-beating,a widely confirmed method for extracting the total DNA from feces,and then we use universal primer 18S rDNA PCR to detect fungi DNA,meanwhile,random cloning and sequence analysis was also demonstrated the quality of fungal DNA extracts using the above method.And the result of this method is also compared with the fungal isolation and culture identification methods Futher more,on the basis of the above, establishment of phylogenetic trees by cloning and sequencing and ERIC-PCR fingerprint map were utilized to analyze the polymorphism of fungal community in ascites of patients with cirrhosis.
【material and method】
1.Collecting stool specimens of 28 Cirrhotic Patients with Ascites、20 Chronic hepatitis B patients(non-active)、30 healthy subjects.One portion was identified to genus by the fungal isolation and culture identification method,the other portion stored in-80℃within 1 hour.
2.Choosing the QIAamp~(?)DNA Stool Mini Kit combined with bead-beating,a widely confirmed method for extracting the total DNA from feces stored in 80℃,and then we use universal primer 18S rDNA PCR to detect fungi DNA,Randomly selected 23 clones from 8 ascites of patients with cirrhosis to establish of phylogenetic trees,and the result of this method is also compared with the fungal isolation and culture identification method.
3.Using ERIC-PCR fingerprint technique to analysis and compare the diversity of fungal flora in the 9 ascites of patients with cirrhosis、 8 Chronic hepatitis B patients(non-active)and 8 healthy subjects.
【Result】
1.Compaired with the fungal isolation and culture identification method,universal primer 18S rDNA PCR combined with QIA+bead beating(universal primer PCR detection method)is more fastly and sensitively to detect fungal colonization and infection of digestive tract,meanwhile,both of them showed that the detective rate of stool fungi from Ascites of Patients with Cirrhosis is higher than Chronic hepatitis B patients(non-active)and healthy subjects.
2.In 8 ascites of patients with cirrhosis's stool specimens,there were 3 candida spp.found by common fungi culture method.but by establishing phylogenetic trees,there were found 5 candida spp.、one Aspergiilus spp.、one Saccharomyces cerevisiae and one Mortierella spp.
3.ERIC-PCR fingerprint technique suggested that:the polymorphism of gastrointestinal fungal community in ascites of Patients with Cirrhosis was more abundant,compared with the Chronic hepatitis B patients(non-active)and healthy subjects.
【Conclusions】
1.Universal primer PCR detection method could extract high quality fungal total DNA from feces,which is beneficial to study on the diversification of gastrointestinal fungus flora composition.
2.the polymorphism of gastrointestinal fungal community in ascites of Patients with Cirrhosis was relatively rich,and Candida spp.is the dominant fungi.
引文
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