Rapid Detection Of Mucor Infection Based On Real-time PCR

Objective:Mucormycosis is an opportunistic fungal infectious disease,which is usually caused by mucormycoles.In the past,it was also called Zygomycosis.The common pathogens included Rhizopus spp.,Mucor spp.,Lichtheimia spp.,Rhizomucor spp.,Cunninghamella spp.,Apophysomyces spp and Saksenaea spp.etc.Mucormycosis is highly invasive,that is,destructive to human tissues,leading to ischemic necrosis,which has a great risk of death for immunocompromised patients.Early diagnosis and treatment are essential in order to improve patient prognosis.Our experiment objective to establish a real-time PCR method for the detection of common pathogens of mucormycosis,so as to quickly and accurately detect the invasive fungal infection caused by mucormycosis,and provide the basis for the clinical diagnosis and treatment of mucormycosis.This experiment included five kinds of filamentous fungal strains of mucormycosis:Cunninghamella bertholletiae,Rhizomucor miehei,Lichtheimia corymbifera,Mucor circinelloides and Rhizopus oryzae.Methods:1.The standard strains of Cunninghamella bertholletiae,Rhizomucor miehei,Lichtheimia corymbifera,Mucor circinelloides and Rhizopus oryzae were purchased from China General Microbiological Culture Collection Center,and the culture medium was prepared to activate the standard strains preserved by vacuum drying and freeze-drying powder;the DNA of the activated strains was extracted by lysis method and magnetic bead method respectively,then select an efficient DNA extraction method.2.Consulting foreign literatures,we designed four primer and probe sequences(a primer and probe sequence shared by Mucor circinelloides and Rhizopus oryzae),and used four fluorophores with different colors to mark the probe sequence respectively.3.Using the nucleic acid extracted from the standard strain as the template,the real-time PCR reaction system was prepared and amplified under suitable conditions to verify the specificity of primers and probes.4.Optimize the reaction conditions of the established real-time fluorescent PCR method,detect PCR at different anneal:ing temperatures,and explore the annealing temperature for the best amplification effect.5.the sensitivity and specificity of the established real-time PCR methods were tested.In this experiment,the dsDNA concentration was determined by Qubit 4.0.The 10 times of 1ng/μL DNA was diluted to 10-1 to 10-6ng/μL,and the sensitivity of the detection method was explored.The DNA was extracted from bacteria and fungi isolated and preserved in the Microbial Laboratory of our hospital,and the specificity of the detection method was obtained.6.In addition,Matrix dissociation time of flight mass spectrometer was used to detect the standard strains.Results:1.The standard strains cultured for 48 hours.The high concentration of fungal DNA could be obtained by lysis method and magnetic bead method.The pyrolysis method lacke suitable samples.Considering that the magnetic bead method can remove protein and other substances in the sample due to the steps of washing impurities,and reduce the influence of subsequent PCR amplification,the DNA extracted by magnetic bead method was selected as the amplification template and satisfactory "S" shaped amplification curve was obtained.2.Product electrophoresis results were 162 Bp,105 Bp,119 Bp,105 Bp and 105 Bp,which were consistent with the target gene.The Ct value of amplification curve was less than 38,which indicated that the amplification was successful and the primer probe was specific for the nucleic acid of standard strain.3.The minimum detection limit of DNA in real-time PCR was 10-5 ng/μL,and the stability of repeated three times was good.The methods has good specificity for the Scedosporium apiospermum,Candida albicans,Candida Crovis;Escherichia coli,Pseudomonas aeruginosa and Acinetobacter baumannii.4.The colony growth morphology and mass spectrum peak images of five kinds of filamentous fungi were recorded,which laid the foundation for the establishment of mass spectrum database for the diagnosis of filamentous fungi in our laboratory.Conclusion:The magnetic bead method can successfully extract Mucor DNA,which can be used as a template for real-time PCR detection.A rapid real-time PCR method for the detection of Cunninghamella bertholletiae,Rhizomucor miehei,Lichtheimia corymbifera,Mucor circinelloides and Rhizopus oryzae has been successfully established.The four primers and probes have good specificity for standard strains.As four independent single channel real-time PCR detection methods,the qualitative diagnosis of Mucor can be achieved.Its clinical application needs further research.