Detection Of Predominant Fungal Pathogens Based On Nylon Membrane Reverse Hybridization And Polymerase Spiral Reaction

In recent years,due to the widespread use of broad-spectrum antibiotics,immunosuppressants and new chemotherapeutic drugs,the development of organ transplantation,hematopoietic stem cell transplantation technology,the outbreak of infectious diseases and the increase in the population of acquired immune syndrome,the incidence of fungal infections increasing year by year,the fatality rate is high,and it is a serious threat to human life and health,which has attracted widespread attention from the medical community.Due to the lack of specific signs in the early clinical stage of fungal infection,diagnosis is difficult and the best treatment period is easy to miss.Therefore,the establishment of a rapid,specific and sensitive detection method for pathogenic fungi is essential for early diagnosis and timely treatment,reducing patient mortality and improving prognosis.At present,the clinical diagnosis methods for fungal infections are mainly morphological observation,histopathological detection,and serological diagnosis,but they have the disadvantages of time-consuming,strong subjectivity,easy to cause secondary trauma,and high false positive rate.Nucleic acid-based molecular biology diagnostic methods have the advantages of shorter time-consuming,strong specificity and high sensitivity,and therefore have broad application prospects in the early clinical detection of fungal infections.Nylon membrane reverse hybridization（NRH）and polymerase spiral reaction（PSR）are relatively new methods for the identification and diagnosis of pathogenic microorganisms,which have been applied to the detection of certain bacteria and viruses.There are few reports.According to the principle of reverse dot hybridization,NRH can visually observe the reaction results through the hybridization of specific probes and target nucleic acids.It has the advantages of strong specificity,large detection throughput,simple operation,low equipment,etc.,and is suitable for promotion and application in clinical laboratories.PSR is a constant temperature amplification method that requires only a pair of primers,and relies on Bst DNA polymerase to amplify target nucleic acids quickly and efficiently at 65℃,with a short time-consuming and high sensitivity.In this study,the main pathogenic fungi of clinical infections,including Aspergillus fumigatus,Candida albicans,Cryptococcus neoformans,Fusarium solani and Zygomycetes,established NRH and PSR detection methods.1.Methods（1）Established NRH detection method:designed specific probes for Aspergillus fumigatus,Candida albicans,Cryptococcus neoformans,Fusarium solani and Rhizopus oryzae with the ITS region of ribosomal DNA as the target sequence,and detected related pathogenic fungi.（2）Establish PSR detection method:design specific primers for Aspergillus fumigatus PSR usingβ-tubulin as the target gene;design specific primers for Candida albicans PSR using the MTO1 as the target gene;GAPDH was the target gene,and the PSR-specific primers of Cryptococcus neoformans were designed to detect related pathogenic fungi.（3）Using the above two methods,the clinical blood samples and lung tissue samples of simulated infections were tested for Aspergillus fumigatus,Candida albicans,Cryptococcus neoformans,Fusarium solani and Rhizopus oryzae,and the two methods were evaluated and compared.2.Results（1）The established NRH method was used to detect Aspergillus fumigatus,Candida albicans,Cryptococcus neoformans,Fusarium solani and Rhizopus oryzae.Identify the above five pathogenic fungi according to the blue-purple spots on the nylon membrane,the sensitivity can reach 5 pg,and it takes 6-8 h.（2）The established PSR method was used to detect Aspergillus fumigatus,Candida albicans,and Cryptococcus neoformans.It can be different according to the size of the specific ladder band（the smallest band of Aspergillus fumigatus is about 120bp,Candida albicans is about 100 bp,and Cryptococcus neoformans is about 110 bp）;it can also be detected visually（the color of the solution changes from purple to purple）.Blue)or OD reading(OD_650nm is about 0.3)for identification,the sensitivity can reach fg level（Aspergillus fumigatus5 fg,Candida albicans 0.5 fg,Cryptococcus neoformans5 fg）,and it takes 2 h.（3）For simulated clinically infected blood samples and lung tissue samples,the positive detection rate of NRH method is 75%-85%;the positive detection rate of PSR method is 80%-90%.3.ConclusionIn this study,NRH and PSR detection methods were established for the main pathogenic fungi Aspergillus fumigatus,Candida albicans,Cryptococcus neoformans,Fusarium solani,and Rhizopus oryzae.These two methods can specifically identify the above five pathogenic fungi,with high sensitivity,short detection time,large throughput and relatively low cost.According to the characteristics of the method,NRH can be selected for screening of infectious bacteria when there are many samples to be tested,and PSR can be selected for rapid detection when detecting trace DNA in early clinical samples.