Rapid Diagnosis Of Systematic Fungal Infections Based On Multiplex PCR And Microsatellite PCR

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.