Rapid Detection Of Predominant Pathogenic Fungi By LAMP And Nanogold Assisted PCR Methods

Invasive fungal infections(IFI) are an increasingly common complication inimmunocompromised patients recently. The nonspecific clinical symptoms, rapidprogression and high mortality are often associated with IFI. It occurs in most clinicaldepartments and treatment remains limite by scarcity of antifungal efficacy andserious side effects，which makes diagnosis diffcult at the onset. Therefore，it issignificant to establish a rapid, specific method for timely diagnosis. And earlydiagnosis is key to early antifungal therapy, decreasing mortality and improvingpatients’prognosis.Conventional culture and histopathological method are importmant for diagnosisof IFI. However, there are shortcomings such as time-consuming and traumatic ofthose methods. Nucleic acid-based diagnostic methods are now available because ofthe superiority of high sensitivity, specificity and rapidness. These new diagnosticmethods facilitate an early diagnosis of invasive fungal diseases and have broadapplication prospects in early clinical testing. Our study focuses on rapid detection ofpredominant pathogenic fungi by loop-mediated isothermal amplification (LAMP)and nanogold assisted semi-nested PCR(snPCR) method.1. Detection of predominant pathogenic fungi by LAMP methodLAMP is a simple, accurate, inexpensive method of nucleic acid amplification.Over the past decade, it has been widely used in the diagnosis of infections such asviruses, bacteria and parasites, but less used in the diagnosis of fungal infections.In this study, specific primers were designed for the LAMP methods based on therRNA gene ITS (internal transcribed spacer) region of Aspergillus fumigatus andCandida spp.. The conditions, including the reaction temperature, the concentration ofMg2+and Bst DNA polymerase, were optimized to determine the appropriate system.According to the specificity of the ladder bands produced by LAMP reaction,A.fumigatus and Candida spp. could be rapid, specific identified, while other clinical common pathogens no amplification. The sensitivity of LAMP (Aspergillus fumigatus6.0pg; Candida0.3pg) is higher than the conventional PCR by10times. More, thereaction could be completed only within1h. Hence, the established method of LAMPis rapid with high specificity and sensitivity. Besides, specimens of blood and tissues(lung, kidney, liver) from infected mice were confirmed by LAMP and fungal culture.The detection rate of LAMP (70%-90%) was higher than the fungal culture, especiallythe rate of blood specimens. Fungal culture took48h at least to get the positiveidentification, while the detection of LAMP could rapidly and specifically diagnosewith or without fungal infection only within3-4h.2. Detection of Candida species by nanagold assisted snPCRSnPCR is a rapid nucleic acid amplication method with high specificity andsensitivity. In this study, specific primers were designed for the this method based onthe GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene of Candida spp..Butthe snPCR is affected by many factors such as the primer, concentration of dNTP,quality of DNA and so on, which resulted in non-specific amplification.So, we added gold nanoparticles(AuNPs) into the snPCR system. The resultsshowed that both the specificity and yield of the snPCR increased after added4nMAuNPs (13nm), indicating AuNPs could eliminate the nonspecific amplication andincrease the yield. The sensitivity of the reaction was up10times higher than thesnPCR without AuNPs, indicating AuNPs could enhance the snPCR effciency. Toamplify the DNA sequence of common clinical pathogens using nanogold assistedsnPCR, only Candida spp. could produce a specific band of c.a.360bp, noamplification of other common clinical pathogens, with high sensitivity up to30fg.Detecting the blood and tissue specimens of infected mice by this method, thedetection rate was between80%and90%, higher than the fungal culture, and thedetection could be completed within6h.In conclusion，the LAMP and nanogold assisted snPCR were successfullyestablished in the study. It comfirmed these methods were a rapid, culture independentdiagnostic technique with high specificity and sensitivity and have potentialapplication in the early clinical testing.