Rapid Diagnosis Of Systematic Fungal Infections Of Experimental Animal Model

In rescent years, the incidence of systematic fungal infection is increasing and the spectrum of pathogens has expanded. Candida, Aspergillus and Zygomycetes (mainly Mucor and Rhizopus) are the most important pathogens of systemic fungal infection.The systematic fungal infections caused by yeasts or filamentous fungi proceed rapidly in the high-risk groups, such as organ transplantation, stem cell transplantation and blood tumor patients. And the diagnosis delayed or misdiagnosis will make the opptunity of treatment lost, which can increase the mortality of patients significantly.The diagnosis methods of systematic fungal infections include culture-based and non-culture-based approaches. Culture-based method is time-consuming, with low positive rate and easy to contaminated. Among non-culture-based methods, there are pathological examination, serological testing, iconography detection, molecular biological diagnosis and so on.In the past studies, Mutiplex PCR technique has been applied in the detection of Candida, Aspergillus and other fungi in blood samples. However, there hasn't been reports about the detection of multiple pathogenic fungi in blood and tissue at the same time by multiplex PCR. We selected clinical isolates (Candida, Aspergillus, Mucor), established experimental animal model of systemic fungal infections, amplified TEF gene,studied the detection efficiency of multiplex PCR in infected blood and tissue, and compared that with positive rates.In order to study conveniently, the experiment has been devided into two parts: In the first part,firstly, three pairs of genus specific primer were designed to distinguish Candida, Aspergillus, Mucor and Rhizopus according to TEF1αprotein coding gene, genus versatility of each primer was detected by PCR; secondly, other fifteen fungi were amplified using the three pairs of primer to evaluate the specificity; finaly, the three pairs of primer were unified to amplify by multiplex PCR.In the second part, according to literature abroad, we selected ICR mice, suppressed their immunity by Adenosine Cyclophosphate peritoneal injection, made experimental mice model of systemic fungal infections, and observed their infection characteristic. On the basis of the first part, the blood and tissue(liver, spleen, lung, kidney)of infected mice were collected to extract nucleonic acid, the mice pathogens were detected by multiplex PCR, and compared with cultivation of blood and tissue(liver, spleen, lung, kidney) samples.Nearly twenty fungi including Candida, Aspergillus, Mucor and Rhizopus were amplified using three pairs of genus specific primer designed by myself, each genus resulted in about 200 bp, 400 bp, 100 bp and 500 bp fragment, there was not cross reaction in and outside the four genera, showing fine specificity. Multiplex PCR could make amplifying the four genera fungi at the same system and time come true. After establishment of experimental animal model, pathogenic fungi could be detected from infected blood and tissue using multiplex PCR. With regard to blood samples, the sensitivity of multiplex PCR was higher than that of cultivation; With regard to tissue samples, the sensitivity of multiplex PCR was no higher than that of cultivation,but the detection time of former was much shorter than latter. The study will continue to approach the identification and diagnosis methods to raise the positive rate of pathogen detection.