The Establishment Of Real-time Fluorescent Quantitative PCR Assays For The Detection Of Aspergillus

Objective:The experiment was carried out to establish real-time fluorescent quantitative polymerase chain reaction(FQ-PCR) assay for detection and strain identification of aspergillus rapidly, accurately and specificity, which is common of invasive fungal infections. Aspergillus mainly includes aspergillus fumigates, aspergillus flavus, aspergillus terreus and aspergillus niger in this experiment. Provide a basis for domestic Aspergillus test kit development. Methods:The standard fungal strains were obtained from CGMCC, and were cultured on the substrate. Cell suspensions were prepared with 0.9% saline and adjusted to about 105 spores /m L, and Fungi solid tissue were weighed approximately 200 mg, which will be used in the establish of the FQ-PCR assay of the fungal tests. Gen Bank(NCBI) was searched for sequences of the genes of lots of fungi species, bacteria, virus and human. At the end verified the specific regions each of those four species. Specific primers and probes for those fungi were designed by the Primer Premier 5.0 and Beacon Designer 7.7, and detected by PCR to filter the most suitable primer and probe. Two different DNA Isolation methods were compared to choose the best fungal DNA isolation method which can be used in the clinical. Those methods contain one-step-heat method and liquid nitrogen grinding method, which compared by extraction efficiency and PCR amplification effect of aspergillus DNA. Specific experiments, repeatability experiments and sensitivity analysis were taken out in each of those four PCR reactions. Results:Aspergillus grow well when was inoculated in SDA culture medium. Different colony morphologies can be observed when grown for 3-4 days at 37 ° C incubator. One-step-heat method and liquid nitrogen grinding method of extraction efficiency are compared by real-time PCR amplification effect of aspergillus DNA, both fluorescence curve present a sigmoid appearance and the Ct value was ahead. Analyze Ct value of the two different extraction methods by two-sample t test, P> 0.05, not statistically significant. Four aspergillus’ primers and probes were designed successfully. Each of the fpur PCR reactions would go on well with specific amplification but without cross-reaction among the other fungi, bacteria, viruses and human gens. The sensitivity of aspergillus fumigates, aspergillus flavus, aspergillus terreus and aspergillus niger FQ-PCR reaction will achieve 35 spores/m L with the good repeatability and stability. All of the real-time protocols tested were highly reproducible, specific and sensitive. The coefficient of variation of Ct value in each Repetitive experimental was less than 5%. Conclusions:1. For fungal solid tissues, liquid nitrogen grinding method of extraction efficiency is high concentration and purity. One-step-heat method is a simple and rapid method of aspergillus’ DNA isolation which was suitable for the FQ-PCR reaction. The FQ-PCR reactions would take on with good result in the condition of that DNA isolation method.2. The establishment of those FQ-PCR assays was based on the success of primer and probe designing. Each assay was a useful tool for the rapid, accurate identification of aspergillus and can give each aspergillus a classification and quantification besides. All of the FQ-PCR reactions were repeatability and stability.