| Objective:(1)In this study,based on the technology of Recombinase Polymerase Amplification(RPA),We constructed rapid detection methods for Candida albicans and optimizes its application.We hope that these methods can provide technical support for the on-site detection of Candida albicans and provide new feasible solutions for the diagnosis and control of Candida albicans.(2)In experiment one,we constructed real-time fluorescent RPA to rapidly detect Candida albicans standard strains and clinical isolates.The purpose of the test is to evaluate the clinical applicability of the method.(3)In experiment two,in order to further promote the application of this technology to bedside,grassroots and even home detection,a lateral flow strip detection(LFD)combination was constructed to meet the needs of Point-of-care testing(POCT)detection of Candida albicans The rapid detection method for recombinase polymerase amplification(LFD-RPA)realizes easy-to-answer of test results and simplifies the interpretation of test results.Methods:(1)In experiment one,at first,we selected the Candida albicans nucleic acid sequence conserved region ITS2 as the identification gene,then designed a series of primers and probes in combination with the guide of the exo kit to select the most suitable primers for subsequent experiments through a primer screening experiment;Secondly,we took a standard Candida albicans strain,using 5 non-Candida albicans strains as the negative control such as Pseudomonas aeruginosa,Staphylococcus aureus,Candida glabrata etc.Then we extracted DNA as templates which we detected by PCR,qPCR and real-time fluorescent RPA.The detection results were sent for sequencing and compared at NCBI.Three methods were used for detecting the specificity of Candida albicans and counting the total time of detection respectively;We took a standard Candida albicans strain for resuscitation,shaking,and extracting the genome.PCR was used to obtain the target fragment to construct a plasmid standard.The concentration was measured and the corresponding copy number/?L was calculated.Seven samples with a concentration of 10~7 to 10~1 copies/?L were tested using the three methods described above.Candida glabrata was used as the negative control.Each gradient was set at least 3 times.At the same time,this method was used to analysis the sensitivity of Candida albicans;Finally,the standard strain of Candida albicans was used as a positive control.The five negative control strains were the same as described above.DNA was extracted by different methods using a genomic kit,heating and boiling,and repeatedly freeze-thawing with liquid nitrogen method.clinical samples of candida albicans were used for real-time fluorescent RPA detection,using the PCR test results as a reference,and checking whether the crude DNA extraction method was feasible.(2)In experiment two,firstly,ITS2 was also selected as the identification gene.With reference to the description of the lateral flow chromatography test strip and the nfo kit design guide,the optimal primers and probe screened for the above-mentioned exo kit were selected.The primers and probe were slightly modified to be used by this experiment;Secondly,we took 1standard Candida albicans strain and 5 strains as the negative control bacteria(same as above),and explored the suitable reaction temperature and detection time,at the same time we analyzed the specificity of the test and calculated the test time;The 7 plasmid standards successfully constructed above were used for experiments and each gradient was set at least 3 replicates.The lower limit of detection was statistically analyzed for detecting the sensitivity of Candida albicans;Finally,clinical samples of Candida albicans were tested and Candida glabrata was used as a negative control that was compared with the detection results of PCR and real-time fluorescent RPA detection methods to determine whether they were consistent.Results:(1)In all three methods,the Candida albicans showed positive results,and none of the negative control showed such ones.The amplified results were sent for sequencing and successfully compared in NCBI.The total time of PCR,qPCR,and real-time fluorescent RPA was respectively 133,78 and 35 min;The detection limits of all tests were 10~1 copies/?L.As the concentration of the plasmid standard increased,the fewer cycles required for the curve to reach the threshold line,the shorter the time required to detect a positive result.The real-time fluorescent RPA test results showed that the concentration gradient of the plasmid standard and the detection time was significantly negatively correlated(R~2=0.91,P<0.01),this experiment is repeatable;The positive detection rate of real-time fluorescent RPA on 31 clinical samples suspected to be infected with Candida albicans was 32.26%(10/31),which was slightly lower than 35.48%(11/31)of PCR.10 samples were successfully identified by both methods.RPA and PCR show good specificity for clinical samples detection.DNA was extracted using the kit and heating boiling method for RPA,and the time required for critical positive amplification were(438±13),(462±12)s,which were similar(t=1.32,P>0.05).The liquid nitrogen was repeatedly frozen and thawed to extract DNA for real-time fluorescent RPA and the required time for the positive amplification was(584±15)s,which was obviously more than the other two methods(t=7.55,6.39,P<0.01).It is feasible to use the crude samples as the detection objects.(2)In the detection of LFD-RPA,we found that no control bands appeared except for Candida albicans and the total detection time was 30minutes;The optimal reaction temperature for this experiment was 37?and the optimal reaction time was 10 minutes.The positive detection rate of 31 clinical samples suspected to be infected with Candida albicans was 45.16%(14/31).Compared with the LFD-RPA's clinical results of Candida albicans,the positive coincidence rates of PCR and real-time fluorescent RPA were 84.62%,76.92%.Conclusion:(1)We had successfully constructed and optimized real-time fluorescent quantitative RPA for detection of Candida albicans,which had the advantages of fast detection,simple process operation,high sensitivity and good specificity.The volume of the reaction system was reduced to approximately 1/3of the recommended amount.In addition,this experiment combined with the crude nucleic acid extraction methods could still perform stably and well,which greatly reduced the costs and time compared with PCR and qPCR.It could meet the high-throughput rapid detection of large-scale samples that usually needed a fluorescence detection device when testing and more suitable for the application of qualified laboratory teams and clinical testing centers;(2)On the basis of real-time fluorescence quantitative RPA,we had further optimized the detection of Candida albicans by LFD-RPA,which could achieve complete detachment from the detection facilities.The experiment only requires a water bath or even body temperature to provide the required temperature.The results of tests were both specific and sensitive,which could be interpreted directly with the naked eye.It is more suitable for untrained personnel to operate that could be popularized and applied in point-of-care testing(POCT)and resource-constrained environments. |
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