Establishment And Evaluation Of Detection For Pathogenic Microorganisms Based On Nucleic Acid Amplification

In recent years,with the continuous outbreak of new infectious diseases,pathogens in the environment have more and more impact on human health.Hospital environment is also one of the main ways of pathogen transmission.Nosocomial infection,also known as a hospital-acquired infection,is an infection whose development is favored by a hospital environment,such as one acquired by a patient during a hospital visit or one developing among hospital staff.The incidence of nosocomial infection is mainly related to invasive operation,abuse of antibiotics,loose aseptic operation,low immunity and so on.Therefore,it is always the focus of nosocomial infection prevention and control work to strengthen the supervision and inspection of pathogenic bacteria in hospital environment.Bloodstream infection（BSI）is one of the nosocomial infection with high incidence in recent years,so it is very important to strengthen its detection and supervision.Currently,blood culture method is the gold standard for clinical diagnosis of BSI,but it has shortcomings such as long etiological detection time and long response time,which can not meet the"rapid diagnosis"required by the diagnosis and treatment of BSI patients.Therefore,it is very important to establish a method that can quickly identify and sensitively detect pathogenic bacteria from clinical whole blood samples at the early stage of infection to prevent and control infection.It is also important to ensure the health of the population.Based on the loop-mediated isothermal amplification technology,gene editing technology,magnetic separation technology,multiplex PCR technology,digital PCR technology and capillary electrophoresis technology,this study takes common BSI in hospital environment as the research object,and aims to construct a rapid,accurate,qualitative and quantitative detection technology for one to multiple bacteria in blood samples through reasonable fusion of the above technologies.These provided new ideas and technical support for hospital environment BSI control.PartⅠEstablishment and evaluation of detection method for four pathogens in bloodstream infection based on MBs-MLT-m PCR-CE techniqueObjective:In this study,a high-throughput method for the detection of four common pathogens（Staphylococcus aureus,Enterococcus faecalis,Escherichia coli,Klebsiella pneumoniae）in clinical blood samples was established by using the antimicrobial melittin functionalized magnetic beads（MBs-MLT）combined with multiplex polymerase chain reaction（m PCR）and capillary electrophoresis（CE）.Methods:Magnetic beads（MBs）with carboxyl group on the surface were synthesized by solvothermal method.MBs-MLT was prepared by coupling antimicrobial peptides capable of non-specific bacterial binding via the amidation of EDC and NHS.Transmission Electron Microscope,Fourier transform Infrared Spectroscopy,hysteresis loop and Dynamic Light Scattering were used to characterize the prepared MBs and MBs-MLT.Staphylococcus aureus was selected as the target bacteria.MBs-MLT was used for magnetic separation and the amount of MBs-MLT captured bacteria was optimized.Then,the enrichment efficiency of MBs-MLT to four common clinical bloodstream infections was determined.Nuc gene of Staphylococcus aureus,16S r RNA gene of Enterococcus faecalis,uid A gene of Escherichia coli,Rob gene of Klebsiella pneumoniae were selected for primer screening.Then some key factors such as primer concentration,d NTPs,Mg²⁺,Hot Start DNA polymerase concentration and annealing temperature were optimized to establish the multiplex PCR system.Four target gene specific primers were modified with FAM fluorophore.After multiple PCR amplification,the target DNA fragments were separated by capillary electrophoresis.The capillary electrophoresis spectrogram and data were collected and processed using Genemapper V5.0.Simulated blood samples were detected and evaluated based on MBs-MLT-m PCR-CE technology.Clinical specimens of bloodstream infection were detected based on MBs-MLT-m PCR-CE.Results:1.TEM,FTIR,DLS and hysteresis loop were used to characterize the synthesized MBs and MBs-MLT.The results showed that the antimicrobial peptides were successfully coupled to the MBs.The enrichment efficiency of the synthesized MBs-MLT to the four target bacteria reached more than 80%.2.The m PCR reaction system was optimized.The reaction system consisted of10μL of 5×PCR buffer,6μL of Mg²⁺（25m M）,4.5μL of d NTPs,0.6μL of Hot Start DNA polymerase（5 U/μL）,and 2.4μL of（10μM）upstream and downstream primers of Staphylococcus aureus,2.4μL of（10μM）upstream and downstream primers of Klebsiella pneumoniae,2.0μL of（10μM）upstream and downstream primers of Enterococcus faecalis,1.6μL of（10μM）upstream and downstream primers of Escherichia coli.The final volume was 50μL.The reaction conditions were predenaturation at 95℃for 5 min,denaturation at 95℃for 20 s,annealing at 55℃for 45 s,renaturation at 72℃for 45 s（35 cycles）,extension at 72℃for 1 min.3.Acinetobacter Baumannii,Pseudomonas aeruginosa,Staphylococcus epidermidis and Streptococcus pneumoniae were selected as specific controls.The m PCR products were detected by agarose gel electrophoresis and capillary electrophoresis.The fluorescence values of target strains were all higher than those of blank strains,and no peak appeared in non-target strains.The results showed that MBs-MLT-m PCR-CE had good specificity.4.Simulated blood samples with different bacterial concentrations（10⁰-10⁵CFU/m L）were prepared.The results showed that the detection limit of MBs-MLT-m PCR-CE was 10 CFU/m L,which was obviously lower than that of ordinary PCR method（10²-10³ CFU/m L）.5.100 clinical samples of suspected blood infections were tested.The overall clinical sensitivity was 90%and the clinical specificity was 100%.The detection time was 3.5 hours,which was significantly shorter than that of traditional blood culture methods.Conclusions:1.In this study,the antimicrobial peptide-functionalized magnetic beads were successfully prepared,which can non-specifically capture four common bloodstream infection bacteria（Staphylococcus aureus,Enterococcus faecalis,Escherichia coli,Klebsiella pneumoniae）with high capture efficiency.2.The reaction conditions of the detection system were optimized,including primers,Mg²⁺,d NTPs,Hot Start DNA polymerase concentration and annealing temperature.The MBs-MLT-m PCR-CE method was established to realize the rapid detection of four kinds of bloodstream infection pathogens.3.The method does not require pregrowth and can be applied to the detection of whole blood bacterial infection,which has great clinical application potential.PartⅡEstablishment and evaluation of detection method for four pathogenic bacteria of bloodstream infection based on MBs@Si O2-NH2-dd PCR technique Objective:In this study,a high-throughput method for the quantitative detection of four common pathogens（Staphylococcus aureus,Enterococcus faecalis,Escherichia coli,Klebsiella pneumoniae）in clinical blood samples was established by the preparation of polyamino-functionalized silicon coated magnetic beads（MBs@Si O₂-NH₂）combined with droplet digital PCR（dd PCR）technology.Methods:MBs were synthesized by improved solvothermal method,and then MBs@Si O₂-NH₂ were synthesized by one-pot method.The prepared MBs and MBs@Si O₂-NH₂ were characterized by TEM,FTIR,DLS and hysteresis loop.MBs@Si O₂-NH₂ was used to capture bacteria and the amount of MBs@Si O₂-NH₂was optimized.The enrichmentefficiency of the optimized MBs@Si O₂-NH₂ for four common bacteria in clinical bloodstream infection captured by MBs@Si O₂-NH₂was determined.Four kinds of specific primers and probes were screened,and then the key factors such as probe concentration and annealing temperature were optimized to establish multiple dd PCR system.The MBs@Si O₂-NH₂ capturing bacteria combined with multiple dd PCR system was established.The sensitivity,repeatability and specificity of the four pathogens were detected and evaluated.Simulated blood samples were detected and evaluated based on based on MBs@Si O₂-NH₂-dd PCR technology.Clinical specimens of bloodstream infection were detected based on MBs@Si O₂-NH₂-dd PCR technology.Results:1.The synthesized MBs and MBs@Si O₂-NH₂ were characterized by TEM,FTIR,DLS and hysteresis loop.The results showed that Si O₂ and NH₂ were successfully coated on the MBs,and the enrichment efficiency of the synthesized MBs@Si O₂-NH₂for the four target bacteria reached more than 90%.2.The multiple dd PCR reaction system was optimized.The final volume of 22μL,consisting of 11μL of 2×HQ d PCR Master（probe）,0.5μL each of the four upstream and downstream primers（10μM）each,0.8μL each of four probes（10μM）,2μL of DNA,and the remaining volume is filled with dd H₂O..The reaction conditions were 60℃for 5 min,95℃for 15 min,45 cycles of 95℃for 30 s and56℃for 60 s,the fluorescence signal was recorded d at 60℃for 60 s.3.The established multiple-dd PCR method had good accuracy and repeatability.For the detection limit,the four target bacterial plasmids in the 22μL dd PCR reaction solution could reach 5 copies/reaction,achieving single-copy detection.The specific results showed that the samples containing four kinds of target bacteria had obvious positive signals,while the negative control and other non-target bacteria had no signals,indicating that the detection system had good specificity.4.Simulated blood samples with different bacterial concentrations（10⁰-10⁵ CFU/m L）were prepared.The results showed that the detection limit of MBs@Si O₂-NH₂-dd PCR method was 10 CFU/m L,which was obviously lower than that of q PCR method（10² CFU/m L）.By using digital PCR integrated data analysis software,Staphylococcus aureus can be detected as few as 7.0 copies/reaction,Enterococcus faecalis can be detected as few as 8.6 copies/reaction,Escherichia coli can be detected as few as 9.9 copies/reaction,Klebsiella pneumoniae can be detected as few as 8.1 copies/reaction.5.30 clinical samples of suspected blood infection were tested.The overall clinical sensitivity was 80%and the clinical specificity was 100%.The detection time takes 3 hours,and the quantitative analysis of the target bacteria was achieved.Conclusions:1.The positively charged MBs@Si O₂-NH₂ were successfully prepared,which could non-specifically capture four common bloodstream infection bacteria with high capture efficiency.2.Various reaction conditions of the dd PCR system were optimized,and the MBs@Si O₂-NH₂-dd PCR method was established to realize the absolute quantitative rapid detection of four kinds of bloodstream infection pathogens.3.This established method can quantitatively detect bacteria in the blood of complex samples without relying on standard curve.This has a guiding significance for clinicians to targeted therapy and drug duration and dosage.Part Ⅲ Establishment and evaluation of detection method for Klebsiella pneumoniae based on LAMP amplification and CRISPR-Cas12a technology Objective:In this study,we combined LAMP amplification with high sensitivity and CRISPR-Cas12a with highly specific sequence recognition function,and placed both systems in the same reaction tube.Therefore,a"one-tube"method for the immediate detection of Klebsiella pneumoniae in bloodstream infection was established.Methods:The LAMP detection system of Klebsiella pneumoniae was established.The reaction temperature and Mg²⁺concentration was optimized.The guide RNA was designed based on the PAM sequence on the amplified Klebsiella pneumoniae fragment,and the nonspecific reporter gene probe with fluorescence and quenching function was synthesized.A suitable CRISPR-Cas12a system was constructed based on LAMP.Mg²⁺concentration and probe concentration were optimized."One-tube"system based on LAMP and CRISPR-Cas12a was established.The detection limit,repeatability and specificity of Klebsiella pneumoniae were evaluated.Rapid detection and evaluation of Klebsiella pneumoniae in simulated blood samples was established based on LAMP and CRISPR-Cas12a mediated"one-tube"immediate detection visualization method.The immediate detection method constructed in this part was used to detect and evaluate clinical samples of blood infection with Klebsiella pneumoniae.Results:1.The LAMP amplification system of Klebsiella pneumoniae was established.The optimal Mg²⁺concentration was 6 m M and the optimal reaction temperature was65℃.2.CRISPR-Cas12a system capable of binding to a LAMP amplification system of Klebsiella pneumoniae was constructed.The optimal probe concentration was 5μM and the optimal Mg²⁺concentration was 40 m M.3.LAMP amplification and CRISPR-Cas12a-mediated"one-tube"visual detection system for Klebsiella pneumoniae was successfully established.The method has high sensitivity with a detection limit of 30 copies/μL.The specificity was good,and the detection time was 70 min.4.The detection limit of Klebsiella pneumoniae was confirmed to be 10 CFU/m L by blood simulation test,which was 100 times lower than that of ordinary PCR method（10³ CFU/m L）.5.For 21 clinical blood samples,the positive results of LAMP-CRISPR-Cas12a method were consistent with the results of gene sequencing and electrophoresis.Sensitivity and specificity are all 100%.Conclusions:1.Two different reaction systems of LAMP and CRISPR-Cas12a were placed in the same reaction tube to avoid aerosol pollution.2.By establishing and optimizing the reaction conditions of the detection system,rapid,accurate and highly sensitive qualitative detection of target DNA was realized.3.The LAMP-CRISPR-Cas12a detection system established in this study requires simple equipment,which can meet the field detection and grass-roots applications.