The Construction Of The Ratiometric Fluorescent Method For Detection Of Burkholderia Pseudomallei Based On Pdots And Dual CRISPR/Cas12a Assisted Signal Enhancement

Objective:Melioidosis is a disease with high rate of severity and mortality caused by Burkholderia melioidosis（Bp）.Early diagnosis and treatment are very essential to control the patient’s condition.At present,the main techniques used for the detection of melioidosis include bacterial culture,indirect hemagglutination assay,enzyme-linked immunosorbent assay,polymerase chain reaction and loop-mediated isothermal amplification.The bacterial culture method is time-consuming,and the serological assay is less sensitive to the early infection stage due to the lack of appropriate uniform diagnostic threshold.In addition,nucleic acid detection methods are still limited by sophisticated equipment and the complexity of thermal cycling steps.Thus,it is imperative to develop a rapid,specific and sensitive detection method for Bp.A ratiometric fluorescent method based on Pdots and dual CRISPR/Cas12a assisted signal enhancement was established,which can achieve rapid,specific and sensitive detection of Bp nucleic acid.Methods:The type III secretion system conserved gene（TTS1-orf2）,which can distinguish Bp from other Burkholderia species with high specificity,was used as the target model to design the ratiometric fluorescent biosensor based on Pdots and dual CRISPR/Cas12a assisted signal enhancement.Firstly,PFBT Pdots were synthesized by classical coprecipitation method,where TAMRA-DNA and PFBT Pdots were used as signal reporter moieties.In acidic buffer,TAMRA-DNA was adsorbed on the surface of Pdots to generate fluorescence resonance energy transfer（FRET）,which obtained a ratiometric fluorescent signal of F_Pdots/F_TAMRA.Cas12a enzyme was added to the FRET sensing platform as a recognition tool,and two recognition sites for Cas12a were designed on a single Bp target DNA.Cas12a specifically recognizes and cleaves the double-stranded DNA（ds DNA）of the target,which the trans-cleavage activity of Cas12a would be activated,cleaving TAMRA-DNA continuously and non-specifically.The ratiometric fluorescent signal of F_Pdots/F_TAMRA is enhanced as the amount of TAMRA-DNA adsorbed on the surface of Pdots decreases.The enzymatic activity of Cas12a,the acid resistance of Pdots and TAMRA,the adsorption and mechanism of TAMRA-DNA on Pdots,the key role of HCl and the feasibility of the sensing method were verified by polyacrylamide gel electrophoresis（PAGE）and fluorescence platform.The parameters of the sensor platform were optimized,and the sensitivity and specificity of the sensor and detection ability of simulated actual sample for the proposed method were evaluated under the optimal conditions.We constructed a quenched fluorescent detection platform based on Cas12a,to compared the detection performance of quenched fluorescent sensor and ratiometric fluorescent sensor.Results:（1）In the simultaneous presence of Cas12a,cr RNA1/2 and target DNA,the appearance of the cleavage product of target DNA and the disappearance of disappearance of TAMRA-DNA on the electrophoresis band verify that cis-cleavage and trans-cleavage activities of Cas12a are successfully activated,which was characterized by 15%PAGE electrophoresis analysis.When TAMRA-DNA and PFBT Pdots were added to buffers with different p H values,the fluorescence intensity of TAMRA increased and that of Pdots decreased as the p H decreased,indicating that TAMRA-DNA could be adsorbed on the surface of Pdots and FRET occurred under acidic conditions.After determining the p H of the signal output environment,the presence of Bp target in the reaction would attenuate the FRET efficiency,which lead to the change of ratiometric fluorescent signal.All of the above results verify the successful construction of the sensor.（2）To verify the importance of the design of Cas12a double recognition sites in this strategy,we designed different targets with single recognition site(Bp _SRS)or double recognition site targets(Bp _DRS)in Bp nucleic acid sequences,respectively.The experimental conditions of the two systems of Bp _DRS and Bp _SRS are optimized,and the optimal conditions of the following experimental parameters are determined.In Bp _DRSsystem:The concentration of Cas12a was 50 n M;the concentration ratio of Cas12a to cr RNA was 1:1.5;the concentration of Pdots was 1.4μg/m L;the volume of 0.1M HCl was 3.5μL;the concentration of Pdots was 1.4μg/m L;the reaction time of Cas12a was40 min.In Bp _SRS system:The concentration of Cas12a was 30 n M;the concentration ratio of Cas12a to cr RNA was 1:1.25;the concentration of Pdots was 1.4μg/m L;the volume of 0.1M HCl was 3.0μL;the concentration of Pdots was 1.4μg/m L;the reaction time of Cas12a was 80 min.（3）The performance of the two reaction systems was analyzed under the optimal experimental conditions.For Bp _DRS,the linear equation F_Pdots/F_TAMRA=0.1587 lgcBp _DRS+0.3998（R²=0.9921）was obtained in the detection range of 1 p M～10 n M,and the linear equation of Bp _SRS in the detection range of 5 p M～10 n M was F_Pdots/F_TAMRA=0.2196 lgcBp _SRS+0.2655（R²=0.9990）.The detection limit of both reaction systems was 1 p M.（4）In order to reflect the detection performance of ratiometric fluorescent sensor,this paper constructed TAMRA-BHQ2 quenched fluorescent sensor to conduct sensitivity test of Bp _DRS under optimal experimental conditions.The linear equation obtained by the sensor strategy in the detection range of 50 p M～20 n M was y=47.6017lgc-72.3898（R²=0.9982）,and the detection limit was 23 p M.（5）In this paper,this sensor is used to perform the tests of specificity and recovery on Bp target.In the specificity tests,targets with simultaneous base mismatches at two recognition sites produced signals that were almost identical to the background tube,and base mismatches at a single recognition site could still produce significant signal changes.The recovery experiments were carried out in the simulated actual samples,which showed the 101.4%～109.5%of recovery rates and the 2.6%～4.1%of relative standard deviation.This study demonstrated the excellent sensitivity,specificity,repeatability and practical application potential of the sensor in Bp nucleic acid detection.Conclusions:In this study,a ratiometric fluorescent method based on Pdots and dual CRISPR/Cas12a assisted signal enhancement was successfully constructed,which realized a rapid,simple,specific and sensitive detection method for Bp target ds DNA.This method has good repeatability,specificity and sensitivity.In addition,it also has the following advantages:（1）The adsorption was used to replace the traditional covalent conjugation to construct the TAMRA-Pdots ratiometric fluorescent sensor,which could improve the repeatability and sensitivity of the sensor.（2）The HCl inactivation was used instead of the traditional heating inactivation,which could quickly terminate the reaction under the circumstance without heating equipment.（3）The design of Cas12a double recognition site increases the trans-cleavage site of Cas12a,which could shorten the detection time and reduce the missed detection caused by Bp genome mutation.It would obtain a detection platform with higher fault tolerance rate.