Detection Of Predominant Fungal Pathogens Based On Real-Time PCR And Nucleic Acids Lateral Flow Strip

In recent years,the incidence and mortality of invasive fungal diseases have increased significantly,posing a serious threat to public health.The use of immunosuppressive drugs,the combination of basic diseases and the invasive diagnosis and treatment of catheters have all increased the risk of infection.In the face of increasingly serious fungal infection,it is very important to establish a rapid,specific and sensitive detection method for pathogenic fungi for early diagnosis and treatment,reducing patient mortality and improving prognosis.Traditional fungal diagnosis methods include direct microscopic examination,fungal culture,serological test and histopathological examination.These methods have been widely used in clinical laboratories,but they all have their own shortcomings.For example,the results of morphological examination are strongly influenced by the operator’s subjectivity;Fungal culture takes a long time,which may delays clinical diagnosis and treatment;Serological detection is easy to produce false positives.Molecular biological diagnosis based on nucleic acid has broad application prospects in fungal infection detection because of its advantages of rapid detection,high sensitivity and strong specificity.In this study,real-time PCR and nucleic acid lateral flow test strip detection methods were established to differentiate and diagnose various pathogenic fungi commonly seen in clinic.Real-time PCR includes dye method and probe method.In this experiment,dye method is used to report fluorescence signals,which is lower in cost than probe method.Combined with melting curve analysis,it is a rapid diagnostic method with high sensitivity to identify products according to different Tm values.Taking fungal ITS region as the target sequence,three sets of primers were designed and real-time PCR method was established to identify 10 common pathogenic fungi.The first group identified Candida albicans,Candida parapsilosis and Candida tropicalis.The second group identified Candida albicans,Aspergillus fumigatus,Rhizopus oryzae and Fusarium solani.The third group identified Aspergillus fumigatus,Aspergillus iger,Aspergillus flavus,Aspergillus terreus and Aspergillus nidulans.Four common fungi,three species of common candida and five species of common Aspergillus were identified,and the sensitivity was 60 copies/μL.It has good specificity and does not react with other fungi or bacteria,and has good repeatability of the results.Nucleic acids lateral flow strip,also known as nucleic acid lateral flow chromatographic sensor,combines traditional PCR technology with antigen-antibody reaction,and uses gold nanoparticles as colorimetric signal to detect the target,so that the reaction results can be observed with naked eyes,and the operation is simple.Taking β-tubulin as the target gene,specific primers were designed,and a lateral flow test strip method for detecting Aspergillus fumigatus nucleic acid was established,with sensitivity of fg level,it has good specificity and does not react with other fungi or bacteria.The two methods established in this study have their respective advantages and are both suitable for rapid diagnosis of fungi.Nucleic acid side flow test strips can be popularized in grassroots laboratories,with simple operation,convenient observation,and low cost;Fluorescent quantitative PCR is selected for detection when sensitivity requirements are high and there may be multiple fungal mixed infections.