Application Of Loop-mediated Isothermal Amplification Technique For Detection Of Trichosporon Asahii

Research background Trichosporon asahii(T.asahii) is a kind of conditional pathogenic fungi which widely exist in nature, in immune deficiency and immunosuppressive or immunocompromised hosts can lead to skin or disseminated infection, especially in the organ transplantation, AIDS or cancer patients are more esay to be effected. In recent years, disseminated infection reports also gradually are increasing in normal immune function groups. The early diagnosis of disseminated trichosporonosis is difficult, this disease is very dangerous, and always infect multiple organs, often accompanied by acute progressive respiratory failure, kidney failure, the symptom such as disseminated intravascular coagulation with poorer prognosis. traditional diagnosis methods of T.asahii, such as pathology, fungal microscopic examination and culture are time-consuming and low sensitivity, which are difficult to meet the clinical needs. So a number of studies devoted to the development conveniently, short time, accurate and objective diagnostic methods, including antigen detection, imaging examination, in situ hybridization and PCR diagnosis. Antigen detection, imaging examination of them have been approved for use in the diagnosis of invasive fungal disease, but they have been used in disseminated trichosporonosis. In recent years, domestic and foreign researchers generally used molecular biology techniques for detecting trichosporon infection. They have higher sensitivity, shorter time and could identify species of pathogenic fungi accurately, some people agree that molecular biology techniques are the gold standard in the diagnosis of fungal infection. Laboratory conventional molecular diagnostic methods mainly included traditional PCR technology, real-time PCR and nested PCR technology, etc.As the rapid development of the molecular biology, the new identification technique based on polymerase chain reaction(PCR) technology has been described widely. Notomi et al. reported a new DNA loop-mediated amplification method(loop-mediated isothermal amplification of DNA, LAMP). It can be used to amplify specific target DNA sequences with high sensitivity and, the amplification can be obtained in about 60 min with four specific primers and strand displacement DNA polymerase in isothermal conditions(approximately 65°C) eliminating the need for a thermal cycler. LAMP assays were reported to be highly specific, sensitive, rapid, and cost-effective. In addition, the LAMP assay could be carried out in the regular laboratory, with a water bath or heating block. It is a potential and valuable means for clinical sample testing.This study applied loop-mediated isothermal amplification technology, and established a new sassy about T.asahii rapid detection method. This method has higher specificity and sensitivity, and can be applied in clinical specimens, has higher application value and research significance.Research purposes: Apply the 1oop-mediated isothermal amplification(LAMP) specificity for detection of T.asahii. Compare the difference of 1oop-mediated isothermal amplification(LAMP)and traditional PCR method to detect T.asahii.Materials and methods Designed specific primers for IGS1 sequence, and selected 15 strains for test, 14 non-asahii trichosporon strains and 23 strains of other clinical pathogenic fungi as a negative control, applied 1oop-mediated isothermal amplification(LAMP) reaction for extracted fungal DNA, observe amplification results and verify the specificity of the primers based on three different ways such as the naked eye, turbidity meter and product gel electrophoresis. template DNA diluted concentrations Ring mediated by isothermal amplification(1oop-mediated isothermal amplification, LAMP) reaction and traditional PCR experiments, compare two methods of detection specificity differences.Result 1. The results indicate that TA-19 primer is possible used as 1oop-mediated isothermal amplification(LAMP) primer to the detection of T.asahii.The positive reaction showed the amplified bands and positive product reaction color, and the positive curve detected by turbidity meter.2. Detection limit for the LAMP reaction was 9.4pg/μl, while it was 94pg/μl genome copies for the PCR assay. This suggested that the LAMP assay is 10 times more sensitive than the PCR assay.3. The correlation between the LAMP assays and the culture and API-20 biochemical method results of the same tissue samples proved to be coincident.Conclusion1. We successfully detected gene of T. asahii using a loop-mediated isothermal amplification(LAMP) assay Conclusion2. The detection limit of the loop-mediated isothermal amplification(LAMP) assay was more sensitive than the PCR assay.3. The loop-mediated isothermal amplification(LAMP) assay may become useful in detection of clinical samples.