Study On Electrochemical Sensing Method Of Microbial SSU RRNA

Microorganisms with unique physiological and metabolic advantages play an important role in various fields.Rapid,sensitive and reliable detection of microorganisms is essential to ensure human health and public safety.The key of microbial detection is to screen high universal and specific microbial biomarkers,and to develop universal,sensitive and low-cost microbial detection methods.Microbial small subunit ribosomal RNA(SSU rRNA)has become an important biomarker for microbial detection due to its unique structure.Electrochemical biosensors have been developed as potential detection methods to break through the bottleneck of conventional methods due to their unique advantages of simple operation,high sensitivity,low cost and real-time online monitoring.In this paper,we proposed a method of screening partial SSU rRNA for rapid detection of microorganisms,based on this method,partial SSU rRNA sequences which can be used to detect all bacteria and all fungi were obtained.Based on MSPQC sensor,using partial SSU rRNA of the bacteria,the fungi,Staphylococcus aureus and Escherichia coli as biomarker,a series of sensitive piezoelectric biosensor for the bacteria,the fungi,Staphylococcus aureus and Escherichia coli was constructed.This paper mainly includes the following five parts:(1)Study on Universal Biomarker Based on Partial 16S rRNA for Bacterial DetectionThe method for screening partial 16S rRNA and screened seven partial sequences of 16S rRNA as universal biomarker were proposed for detection of bacteria.The universality,specificity and hybridization efficiency were used as criteria in screening method.Seven partial 16S rRNA sequences which screened out by proposed method were of characters of universality,specificity and hybridization efficiency.Using selected biomarker,fifteen pathogenic bacteria in clinical samples were detected without cultivation.The detection limit was as low as 1 CFU/m L.The sensitivity and specificity of the method using proposed sequences as biomarker were evaluated by plate counting method,which was 89%and 95%,which meet the clinical requirement.Furthermore,reverse complementary sequences of them can also be used as primer and probe applied in various nucleic acids hybridization techniques and biosensors,making them promising applications in a broad range of areas.(2)Study on Universal Biomarker Based on Partial 18S rRNA for fungal DetectionThe method for screening partial 18S rRNA and screened five partial sequences of 18S rRNA as universal biomarker were proposed for fungal detection.The universality,specificity and hybridization efficiency were used as criteria in screening method.Seven partial 18S rRNA sequences which screened out by proposed method were of characters of universality,specificity and hybridization efficiency.Using selected biomarker,five pathogenic fungi were detected without cultivation.Furthermore,reverse complementary sequences of them can also be used as primer and probe applied in various nucleic acids hybridization techniques and biosensors,making them promising applications in a broad range of areas.(3)An unltra-sensitive electrochemical sensor targeting screened universal bacterial sequences based on DNA nanomachinesAseptic humoral infection has the characteristics of high mortality and high morbidity,which causes serious health and economic losses to individuals and society.However,the lack of a rapid,reliable and simple method for bacterial detection affects the timely diagnosis and treatment of aseptic humoral infection.In this paper,we constructed an unltra-sensitive bacterial detection method based on target-recycle DNA nanomachine.Using screened partial 16S rRNA as bacterial biomarker,Mycobacterium tuberculosis in blood sample were detected by the constructed bacterial detection method.The detection limit was as low as 40 CFU/m L,and the detection time was 35 min.Compared with the culture method,the sensitivity of the proposed method was 85%(17/20),and the specificity was 89%(40/45).This method only needs two steps to realize the rapid detection of bacteria without complicated pretreatment such as bacterial culture and nucleic acid purification.The proposed bacterial detection method has broad application prospect in clinical detection.(4)A supersensitive MSPQC sensor targeting specific sequences of E.coli based on 16S rRNA and“gene switch”The early detection of bacterium plays a significant role in addressing serious public health issues.In this paper,a supersensitive multichannel series piezoelectric quartz crystal(MSPQC)sensor of bacterium based on 16S rRNA and“gene switch”was constructed.The fragment in specific region of 16S rRNA was used as the biomarker of bacterium to ensure specificity and achieve the accurate judgment of microbial vitality.“Gene switch”was designed to conduct two electrodes by switching insulated“gene-link”into conductive“metal-link”,which achieved the super-sensitivity of MSPQC to bacteria.To demonstrate the feasibility of this strategy,a proof-of-concept method for Escherichia coli assay was designed.The detection limit was down to 2 CFU/m L.Staphylococcus aureus,Salmonella enteritidis,Listeria innocua and Pseudomonas aeruginosa did not interfere with the detection results.Proposed method was highly sensitive,and specific for bacterium detection,which might find widely use in early detection of bacterium in the field of public safety monitoring and clinical diagnosis.(5)Electrochemical biosensor targeting specific sequences of Staph.aureus based on facile synthesis of silver wire across electrodesThe early detection of bacteria is of critical importance in addressing serious public health problems.Here,an electrochemical biosensor for rapid detection of bacteria based on facile synthesis of silver wire across electrodes was constructed.High-variable region of 16S rRNA of bacteria was used as biomarker.Polymerase-free synthesis of silver wire was introduced into electrochemical signal transduction to improve the sensitivity of electrochemical detection.The construction biosensor of proposed method is as follows:Metastable hairpin probe H1 was modified on electrode surface,biomarker can open the stem-loop structure of H1 and activates HCR.The alternate opening of the stem-loop structure of H1 and H2-Au NPs finally results in the formation of long double-stranded DNA-RNA(HCR products)-Au NPs.The formed Au NPs modified HCR products were blown in one direction using N₂to across the electrode gap.Using this HCR products as template,the silver wire was formed between the electrodes by silver deposition,and resulted in sharp change in electrical parameters of electrode.As the proof-of-concept work,multichannel series piezoelectric quartz crystal(MSPQC)was utilized as detector.The detection of Staphylococcus aureus in the concentration range from 50 to 10⁷CFU/m L within 100 min was achieved.The detection limit was 50 CFU/m L.Escherichia coli,Salmonella enteritidis,Listeria innocua,Pseudomonas aeruginosa and Streptococcus pneumoniae did not interfere the detection results.This newly proposed electrochemical biosensor is simple,rapid and exhibit high signal-to-noise ratio,it has great potential for being applied in food safety monitoring and clinical diagnosis.