Rapid Detection Of Aspergillus Fumigatus Using Multiple Cross Displacement Amplification Combined With Nanoparticles-Based Lateral Flow

Objective: To detect whether the Multiple Cross Displacement Amplification Combined with Nanoparticles-Based Lateral Flow(MCDA-LFB)detection method is suitable for the rapid,sensitive and specific detection of Aspergillus fumigatus and it can become a new method in clinical laboratories,We hope it can improve the level of clinical diagnosis of aspergillus infection.Methods: A set of 10 primers was designed specifically to recognize 10 different regions of the Aspergillus fumigatus-specific gene annexin ANXC4.The lateral flow biosensors(LFB)have been devised to visually detect double-labeled MCDA amplification within 1 to 2 minutes.We collected Aspergillus fumigatus strains and non-Aspergillus fumigatus strains,one of Aspergillus fumigatus was used as reference strain to optimize the MCDA-LFB assay.The appropriate temperature and time sensitivity and specificity of the MCDA-LFB assay was evaluated,and we tested 100 sputum specimens with high suspicion of fungal infection using culture,PCR and MCDA-LFB assay.And finally,we compared the results of MCDA-LFB method with those of the culture and PCR methods.Results: The best MCDA condition was 66℃ for 35 minutes,the minimum concentration that can be detected by this method was 10 fg.No cross-reactivity was shown and the specificity of this assay is 100%.We found that the whole process including the preparation of DNA,MCDA reaction and results reporting.The whole process could be controlled within 40 min including MCDA reaction and results reporting.In the case of 100 sputum samples,20(20%)and 15(15%)samples were positive by MCDA-LFB and PCR method,respectively.MCDA-LFB and traditional culture method showed the same results.Compared with the culture,the diagnostic accuracy of MCDA-LFB can reach 100%.These results showed that the MCDA-LFB method has better detection ability than the PCR method.Conclusion: The MCDA-LFB detection method is suitable for the rapid,sensitive and specific detection of Aspergillus fumigatus and it provides a new detection method for clinical diagnosis of aspergillus fumigatus infection...