Establishment And Evaluation Of Rapid Detection Method For Sporothrix Globosa In The Environment

Sporothrix globosa is the main endemic strain causing sporotrichosis.This fungus exists widely in the environment.It is mainly saprophytic in soil or plants with hyphal form.Presence of S.globosa poses a great threat to the health of farmers and gardeners engaged in agriculture or forestry.So,rapid and accurate detection of S.globosa in the environment is of great significance for monitoring the distribution of pathogenic fungus,controlling the spread of diseases,clarifying the mode of transmission,protecting high-risk populations and avoiding outbreaks.At present,the detection of S.globosa is mainly carried out in laboratory.The common methods include isolation culture method and biochemical identification method.These methods usually have limitations such as low sensitivity,poor specificity and time-consuming.Therefore,it is necessary to establish a sensitive,specific,convenient and efficient method for the detection of S.globosa.Objective:The aim of this study was to establish a new method for rapid detection of S.globosa in the environment by combining immunoglobulin of yolk（IgY）,immunomagnetic separation and loop mediated isothermal amplification（LAMP）.The sensitivity,specificity and stability of this method were evaluated systematically,providing certain theoretical and technical support for pathogen detection in the environmental.Methods:1.Preparation and characterization of IgYTwo different antigens were prepared from S.globosa conidia suspension:formaldehyde inactivated antigen and ultrasonic crushing antigen.The vaccines were prepared by mixing the Frank-type adjuvant and antigen in equal proportion to immunize high-yielding hens with specefic pathogen free（SPF）status and common status.Eggs were collected before and after immunization.IgY was extracted by polyethylene glycol precipitation method.BCA protein quantitative kit was used to determine the protein concentration of the extract.Indirect Enzyme-Linked Immunosorbent assay（Indirect ELISA）was optimized to determine the titer of IgY and verify the specificity of IgY.The molecular weight of IgY was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）.2.Preparation,optimization and evaluation of immunomagnetic beadsUsing 1-（3-Dimethylaminopropyl）-3-ethylcarbodiimide hydrochloride（EDC·HCl）and N-Hydroxysuccinimide（NHS）as activators,carboxy-modified magnetic beads were combined with anti-S.globosa IgY to prepare immunomagnetic beads by direct coupling method.The optimal enrichment efficiency was achieved by systematic optimization of the amount of immunomagnetic beads.3.Establishment and optimization and of LAMP methodThe database was consulted to search the specific conserved genes of S.globosa.The LAMP primers were designed and screened.Candida albicans and Aspergillus fumigatus DNA were used to verify the specificity of the screened primers.The key factors were further optimized to establish LAMP amplification system for S.globosa.4.Establishment and evaluation of the rapid detection method for S.globosaThe S.globosa conidia were isolated and enriched by the above developed immunomagnetic beads.The captured S.globosa genomic DNA was extracted by boiling method.The target gene amplification and signal amplification were achieved by LAMP reaction.The proposed method was applied to the detection of soil mock contaminated samples and corn straw mock contaminated samples.The sensitivity and stability of the method were evaluated systematically.Results:1.Preparation and identification of IgYThe results showed that the IgY titer of SPF hens immunized with formaldehyde inactivated antigen was the highest,which was 1:16000.Indirect ELISA results showed that there was no cross-reaction between IgY and non-target bacteria,indicating good specificity of IgY.SDS-PAGE clearly showed the heavy chain and light chain bands of IgY.The average protein concentration of IgY was 7.71±3.87 mg/m L.2.Evaluation result of enrichment effect of immunomagnetic beadsThe results of condition optimization showed that the enrichment efficiency was the highest,which was 81.27%,when the concentration of immunomagnetic beads was0.7 mg/m L.S.globosa were captured at the same concentration using 0.7 mg/m L immunomagnetic beads and 0.7 mg/m L non-immunomodified magnetic beads（BSA-modified magnetic beads）.The enrichment efficiency of immunomagnetic beads was significantly higher than that of BSA-modified magnetic beads.3.Establishment and optimization of LAMP methodThree sets of LAMP primers（ITS,CAL-1 and CAL-2）were designed with the internally transcribed spacer gene（ITS）and calmodulin gene（CAL）as target genes of S.globosa.Real-time amplification results showed that ITS-primers had the best amplification effect.The specificity of ITS-primers was verified by detecting the DNA of non-target fungi.The optimal reaction temperature was 69℃,and the optimal Mg²⁺concentration was 6 m M.The optimized LAMP system achieved target gene amplification within 30 minutes.4.Establishment and evaluation of the rapid detection method for S.globosaUnder the optimal reaction conditions,the detection limit of S.globosa was4.66×10² cells/m L.The proposed method was applied to detect the mock contaminated samples of soil and corn straw,and the target fungus of 4.66×10² cells/m L were accurately detected.The standard curve equations were y=-1.231x+20.427（R²=0.999）,y=-1.661x+23.421（R²=0.995）,respectively.When 100 times concentration of non-target fungi was added into the soil and corn straw mock contaminated samples,S.globosa at low concentration（4.66×10² cells/m L）was accurately detected,indicating that the method had good anti-interference ability.The whole detection process was simple and could be completed within 120 minutes.Conclusion:1.This study prepared anti-S.globosa IgY with high titer,good specificity and application value.The prepared anti-S.globosa IgY was original and innovative,and no relevant reports about anti-S.globosa IgY have been seen at home or abroad.2.In this study,a new detection method based on IgY,immunomagnetic separation and LAMP was successfully established to achieve efficient and convenient detection of S.globosa.3.The method had good specificity,simple operation and strong anti-interference performance.It realized the detection of S.globosa in the environment within 120minutes.The detection limit was 4.66×10² cells/m L.This method has good practical application value.