多重PCR及微卫星标记技术用于深部病原真菌感染的快速诊断
摘要
深部真菌感染的发病率随着免疫受损人群的不断增多呈逐渐上升趋势，已经成为院内感染死亡的主要原因之一。近年来病原菌也发生了菌谱演变，临床上不断出现新型的真菌感染。因此，快速、敏感、特异地鉴定病原菌对于临床真菌感染的诊断和治疗具有重要的意义。
本研究根据真菌翻译延伸因子序列设计种、属特异性引物，或根据全基因组序列设计微卫星引物，以复合PCR、微卫星技术，探讨病原真菌的快速、敏感、特异的鉴定、诊断技术。
利用翻译延伸因子基因属特异引物组合(念珠菌属、曲霉属、隐球菌属组合或曲霉属、毛霉属、镰刀菌属组合)，进行多重PCR均扩增获得属特异性条带。且多重PCR反应可检测出100fg的真菌DNA。因而具有很好的特异性和敏感性。用属特异引物与白色念珠菌种特异引物组合，可获得白色念珠菌种特异条带(537bp)。
根据基因库中已公布的真菌全基因序列设计了四种单一微卫星核心序列引物，利用正交设计优化了反应体系。对烟曲霉可扩增出2～5条带，分子量约在360～1960bp之间。不同引物对烟曲霉的扩增结果有明显差异，而同一种引物可获得清晰的特异性带型。这四种引物对烟曲霉的鉴定均具有种特异性。实验具有良好的重复性和稳定性。
通过本研究建立了主要病原真菌的快速、敏感、特异的鉴定、诊断技术，为临床真菌感染的诊治、预防、流行病学、疾病发生发展规律等研究提供有力的科学依据和技术支撑。
The frequency of invasive opportunistic fungal infections has risen worldwide as a consequence of the rising number of immunocompromised hosts. The varieties and distributions of pathomycete have also changed greatly. Although The predominant cause of fungal infections in hospitalized patients remain, several non-albicans Candida spp. Aspergillus, Mucor, Fusarium, Penicillium, Paecilomyces by opportunistically invasive mycotic infection apparently increases. Therefore, it is important for clinicians to identify the species responsible for the infection. It is quite necessary to find some new diagnostic methods for it.
The aim of this study is to aim to establish a kind of molecular diagnosis assay that could detect and identify the species of pathogenic fungi inducing systemic fungal infectiom accurately, specifically, rapidly and sensitively.
Microsatellite is also named as short tandem repeats (STR) or Simple sequence repeats (SSR). The technology of SSR utilizes microsatellite as a primer to amply by PCR, then identifies based on the feature of lanes. It not only owns stability of DNA fingerprint, but also has the high performance and convenience of RADP. As a result, there are some significance for identify and diagnosis of pathogenic fungi.
chapter one: This paper (chapter one) was to describes a new assay based on TEFla multiplex PCR amplification for direct detection and identification of fungi in clinical specimens. Establish there kinds of specificity primer groups, to detect the major clinical pathogenic fungi respectively.
species-specific primers to identify 44 species of pathogenic fungi inducing deep fungal infection, thus establishing the method of detection and identification. The first result of prime group from multiplex PCR shows that the amplification lengths of Candida, Aspergillus, Cryptococcus are 233bp, 374bp, 147bp respectively. The second result of primer group from multiplex PCR shows that the amplification lengths of Aspergillus, Fusarium, Mucor are 374bp, 131bp, 562bp respectively. The genus universal primers did not amplify any other fungal DNA tested, and PCR was able to detect the presence of as few as 100 fg DNA of fungus. M-PCR could detect and identify the species of pathogenic fungi inducing systemic fungal infectiom accurately, specifically, rapidly and sensitively.
Because Candida albicans remains the most common fungal pathogen, the multiplex PCR method was developed to identify the Candida albicans first from fungal pathogens. The fungal TEF1a gene is multicopy and highly conserved, there are variable regions in it. The sequences of species-specific primers come from variable regions. Candida albicans DNA was amplified with universal fungal primers and specific Candida albicans primers in one reaction tube. Two amplified products were obtained, 233 bp (product of genus universal primers) and 537bp (product of specific primers).
The method was tested against conventional identification on 36 different clinical samples from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but 2 speciment, including Candida, Aspergillus, Cryptococcus, 36, 7, 1 respectively. The results of 2 speciment disagreed with those of culture. Candida albicans DNA was amplified with genus universal fungal primers and specific Candida albicans primers in one reaction tube.
Species identification time was reduced to 1 day with the multiplex PCR. Multiplex PCR assay provides a novel approach for identifying fungi and potential application value in the early diagnosis.
chapter two: This study utilized SSR as a primer for PCR and built a method of detecting and identifying SSR of Aspergillus DNA, which has the features of speediness, specific, sensitiveness and preciseness.
According to the publicized entire gene order of Aspergillus in gene bank, we analyzed and compared with other fungi DNA by software for example BLAST, DNAStar etc. Drawing assistance with computer designing software (Tandem Repeats Finder 3.21), we designed 4 kinds of single primers - 2 kinds of four nucleotides, 1 kind of five nucleotides, and 1 kind of six nucleotides - for PCR reaction to analyze the conservation and specificity. PCR reaction conditions were optimized with orthogonal design from 4 kinds of factors such as Taq DNA, template DNA, dNTP, and primer, at the same time defined the annealing temperature and the cycle numbers.
This study makes use of SSR-PCR, amplifies DNAs of the major disease fungus (Candida, Aspergillus, Fusarium), for the purpose that observe diversities of PCR fingerprint and relationships between genotypes and traditional phenotypes. As the electropherograms show, the most results have 2 to 5 bands, molecular masses between 360to 1960bp. Different primers give rise to apparent diversity. However, the same primer owns the common bands.
After 4 kinds of primers amplifying, Aspergillus show different bands. Comparing different Aspergillus from several sources, there is the duplicate band for certain primer. The results unchanged while repeating experiments. Such nice stability makes statistical result credible, and lead to accumulate the comparability among different genera of fungi. In that, we gain a conclusion that Aspergillus can be distinguished by SSR-PCR, and these 4 kinds of primers are recommendable.
What is stated above, this study provides a significance that is for survey of deep fungal epidemiologic and prevention, on the basis of studying disease fungus genealogical classification, applying microsatellite mark and multiplex PCR technology to analyze the sequences of microsatellite DNA in fungal genome.
本研究根据真菌翻译延伸因子序列设计种、属特异性引物，或根据全基因组序列设计微卫星引物，以复合PCR、微卫星技术，探讨病原真菌的快速、敏感、特异的鉴定、诊断技术。
利用翻译延伸因子基因属特异引物组合(念珠菌属、曲霉属、隐球菌属组合或曲霉属、毛霉属、镰刀菌属组合)，进行多重PCR均扩增获得属特异性条带。且多重PCR反应可检测出100fg的真菌DNA。因而具有很好的特异性和敏感性。用属特异引物与白色念珠菌种特异引物组合，可获得白色念珠菌种特异条带(537bp)。
根据基因库中已公布的真菌全基因序列设计了四种单一微卫星核心序列引物，利用正交设计优化了反应体系。对烟曲霉可扩增出2～5条带，分子量约在360～1960bp之间。不同引物对烟曲霉的扩增结果有明显差异，而同一种引物可获得清晰的特异性带型。这四种引物对烟曲霉的鉴定均具有种特异性。实验具有良好的重复性和稳定性。
通过本研究建立了主要病原真菌的快速、敏感、特异的鉴定、诊断技术，为临床真菌感染的诊治、预防、流行病学、疾病发生发展规律等研究提供有力的科学依据和技术支撑。
The frequency of invasive opportunistic fungal infections has risen worldwide as a consequence of the rising number of immunocompromised hosts. The varieties and distributions of pathomycete have also changed greatly. Although The predominant cause of fungal infections in hospitalized patients remain, several non-albicans Candida spp. Aspergillus, Mucor, Fusarium, Penicillium, Paecilomyces by opportunistically invasive mycotic infection apparently increases. Therefore, it is important for clinicians to identify the species responsible for the infection. It is quite necessary to find some new diagnostic methods for it.
The aim of this study is to aim to establish a kind of molecular diagnosis assay that could detect and identify the species of pathogenic fungi inducing systemic fungal infectiom accurately, specifically, rapidly and sensitively.
Microsatellite is also named as short tandem repeats (STR) or Simple sequence repeats (SSR). The technology of SSR utilizes microsatellite as a primer to amply by PCR, then identifies based on the feature of lanes. It not only owns stability of DNA fingerprint, but also has the high performance and convenience of RADP. As a result, there are some significance for identify and diagnosis of pathogenic fungi.
chapter one: This paper (chapter one) was to describes a new assay based on TEFla multiplex PCR amplification for direct detection and identification of fungi in clinical specimens. Establish there kinds of specificity primer groups, to detect the major clinical pathogenic fungi respectively.
species-specific primers to identify 44 species of pathogenic fungi inducing deep fungal infection, thus establishing the method of detection and identification. The first result of prime group from multiplex PCR shows that the amplification lengths of Candida, Aspergillus, Cryptococcus are 233bp, 374bp, 147bp respectively. The second result of primer group from multiplex PCR shows that the amplification lengths of Aspergillus, Fusarium, Mucor are 374bp, 131bp, 562bp respectively. The genus universal primers did not amplify any other fungal DNA tested, and PCR was able to detect the presence of as few as 100 fg DNA of fungus. M-PCR could detect and identify the species of pathogenic fungi inducing systemic fungal infectiom accurately, specifically, rapidly and sensitively.
Because Candida albicans remains the most common fungal pathogen, the multiplex PCR method was developed to identify the Candida albicans first from fungal pathogens. The fungal TEF1a gene is multicopy and highly conserved, there are variable regions in it. The sequences of species-specific primers come from variable regions. Candida albicans DNA was amplified with universal fungal primers and specific Candida albicans primers in one reaction tube. Two amplified products were obtained, 233 bp (product of genus universal primers) and 537bp (product of specific primers).
The method was tested against conventional identification on 36 different clinical samples from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but 2 speciment, including Candida, Aspergillus, Cryptococcus, 36, 7, 1 respectively. The results of 2 speciment disagreed with those of culture. Candida albicans DNA was amplified with genus universal fungal primers and specific Candida albicans primers in one reaction tube.
Species identification time was reduced to 1 day with the multiplex PCR. Multiplex PCR assay provides a novel approach for identifying fungi and potential application value in the early diagnosis.
chapter two: This study utilized SSR as a primer for PCR and built a method of detecting and identifying SSR of Aspergillus DNA, which has the features of speediness, specific, sensitiveness and preciseness.
According to the publicized entire gene order of Aspergillus in gene bank, we analyzed and compared with other fungi DNA by software for example BLAST, DNAStar etc. Drawing assistance with computer designing software (Tandem Repeats Finder 3.21), we designed 4 kinds of single primers - 2 kinds of four nucleotides, 1 kind of five nucleotides, and 1 kind of six nucleotides - for PCR reaction to analyze the conservation and specificity. PCR reaction conditions were optimized with orthogonal design from 4 kinds of factors such as Taq DNA, template DNA, dNTP, and primer, at the same time defined the annealing temperature and the cycle numbers.
This study makes use of SSR-PCR, amplifies DNAs of the major disease fungus (Candida, Aspergillus, Fusarium), for the purpose that observe diversities of PCR fingerprint and relationships between genotypes and traditional phenotypes. As the electropherograms show, the most results have 2 to 5 bands, molecular masses between 360to 1960bp. Different primers give rise to apparent diversity. However, the same primer owns the common bands.
After 4 kinds of primers amplifying, Aspergillus show different bands. Comparing different Aspergillus from several sources, there is the duplicate band for certain primer. The results unchanged while repeating experiments. Such nice stability makes statistical result credible, and lead to accumulate the comparability among different genera of fungi. In that, we gain a conclusion that Aspergillus can be distinguished by SSR-PCR, and these 4 kinds of primers are recommendable.
What is stated above, this study provides a significance that is for survey of deep fungal epidemiologic and prevention, on the basis of studying disease fungus genealogical classification, applying microsatellite mark and multiplex PCR technology to analyze the sequences of microsatellite DNA in fungal genome.
引文
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