Establishment and Evaluation of an Ultra-rapid Method for the Detection of Orientia tsutsugamushi
Objective:To establish an ultra-fast nucleic acid test for Orientia tsutsugamushi that can be used in grassroots medical institutions.Methods:Using quantitative real-time PCR(qPCR),we compared and analyzed the whole genome sequences of Orientia tsutsugamushi in the database,identified the target genes,designed qPCR-specific primers and probes,optimized the reaction system and reaction procedure,and established a multiplex qPCR ultra-rapid assay.The sensitivity,reproducibility,and minimum detection limit of the method were evaluated using viral nucleic acids quantified by droplet digital PCR(ddPCR)as standards.Comparative genomic analysis was performed to verify the specificity of Orientia tsutsugamushi with pathogens of the same family or genus that tend to cause similar symptoms.Specificity was evaluated using nucleic acids from 24(45 cases in total)other pathogens that tend to cause similar symptoms.The results of the multiplex qPCR ultra-rapid assay and the conventional qPCR assay were compared using 116 clinical whole blood samples by four-grid table chi-square test,and the agreement between methods was analyzed using the Kappa consistency test.Results:Orientia tsutsugamushi has low homology to pathogens of the same family or genus that tend to cause similar symptoms.Multiplex qPCR ultra-rapid assay showed good specificity and no cross-reactivity with 24 common pathogens.The minimum detection limit was 156 copies/mL.Repeatability comparison analysis showed a coefficient of variation of ≤ 3.13％for Ct values for each concentration assay.The differences between the multiplex qPCR ultra-rapid assay and the conventional qPCR assay for simultaneous testing of 116 clinical whole blood samples were not statistically significant(P＞0.05),the two methods had high agreement(Kappa=0.939 2,P＜0.001),with total compliance rate 97.42％(CI:92.67％～99.12％),and the testing time could be be reduced to 30 min.Conclusion:The multiplex qPCR ultra-rapid assay has good sensitivity,reproducibility and specificity,which can rapidly diagnose Orientia tsutsugamushi nucleic acid in clinical whole blood samples and achieve timely and rapid treatment in grassroots medical institutions.
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