| Background:In recent years, infectious diseases are still one of the biggest potential harm to human health, especially in developing countries. The World Health Organization announced that in recent 30 years, 29 new pathogens have been found. C urrently, the species of pathogenic microorganisms which cause human infectious diseases are becoming more and more compicated, common pathogens threat to humans ha ve not been eliminated, there are some new pathogens emerged, which brought great difficulties to the diagnosis and treatment of diseases. A lot of research data show that: with the overuse of antibiotics, not only the burden on patients increased, the number of drug- resistant strains is also becoming more and more, which improve the difficult of treatment of diseases. Therefore, early rapid and accurate identification to pathogenic microorganisms and authentication, can not only use the appropriate antibiotic therapy, and also can improve the prognosis of the patient effectively, which is the most effective way to reduce the mortality. At present the detection and identification of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus aureus and other pathogenic microbes often use the method of isolation and culture combined with the main morphological characteristics, application of biochemical experiment and immune analysis. In clinical practice, the training complex operation, time-consuming, specificity is not strong and other reasons, which can cause false positive or false negative results. These are the deficiency of the current method of pathogenic microorganism detection.With the wide application of molecular biology in the medical field, detection of pathogen genes has become an indispensable method for clin ical diagnosis of disease. And a nucleic acid sequence composed of a large number of pathogenic microorganisms have been obtained and included in the nucleic acid sequence database, which makes the detection of pathogenic microorganism identification metho d on nucleic acid as the goal possible. The biosensor is an interdisciplinary composed of biomolecules(nucleic acids, proteins, biofilms, etc.) and physical chemistry, which is widely used in the laboratory diagnosis and the field of monitoring. Detection principle of the biosensor is a process that the biological reaction signal is converted to easy detection, transmission and processing of electrical signals, and then by the second converted to a digital signal which can be amplified and output. Because of the different converters, the biosensor can be divided into semiconductor biosensor, electrochemical sensors, piezoelectric crystal biosensors and surface plasmon resonance(SPR) biosensors. Among them, the SPR biosensor can be detected by nucleic acid of pathogenic microorganism, but also can achieve the highly parallel detection. SPR is an optical detection technique of reflection and refraction of a light utilization, because the optical device has the advantages of high sensitivity, label free, convenient operation and realtime, fast, online detection etc. In the past years, SPR biosensors have been widely used for the detection of interaction of several biological molecules themselves, including proteins, peptides, nucleic acid, bacteria and viruses. In the SPR biosensor chip, the metal surface binding biomolecules will cause the index and the incident angle changes of SPR, so that the change of the SPR can be monitored to study biomolecular interactions. In order to improve the SPR signal changes more effectively, this study take the solid nano- rolling circle amplification technology, to ampily the SPR signal effectively.Rolling circle amplification(RCA) is a method of nucleic acid amplification at constant temperature, developed in recent years. RCA reaction take the DN A molecular ring as a template, by a short promoter sequence(hybridized and complemented by a part of the annular DNA template molecular), in the Phi 29 DN A set under the catalysis of enzyme to transform d N TPs into a long single strand DNA fragments. The reaction product of RC A contains a large number of repeated DN A template and complementary sequence, so as to realize the amplification of a target nucleic acid, now the RCA technology has been successfully applied to detect different pathogens. Compared with the traditional methods of molecular biology, RCA has a lot of advantages. It does not require a special temperature control instrument and has the ability of linear amplification.Otherwise, RCA products are easy to be fixed on the biochip.In recent years, due to the gold nanoparticles(Au NPs) unique physical and chemical properties and high specific surface area, the Au NPs has attracted more and more attention in the field of biological compatibility test. This is mainly because the metal surface of Au NPs has the strong ability of stable immobilization to biological molecules with thiol group. The Au NPs provides a quick, efficient and high throughput detection platform.It is also increasingly used in SPR biosensor.Therefore, this study on the basis of O ur previous study, based on nano- rolling circle amplification(Nano-RC A) technology, designed six kinds of pathogenic microorganisms lock genomic DNA probe(Padlock Probe, PLP), specifically identifying genomic target sequence. The annular padlock probes connected by a ligase, as an annular template of rolling circle amplification, hybridized with the capture probe immobilized on the surface plasmon resonance(SPR) biosensor chip surface. By this way to establish the biosensor chip surface rolling circle amplification system, reproduce annular lock type probe in situ, and amplified hybridization signal of the sensor surface padlock probe and capture probe. Then using a hybridization of gold amplification contained probe nanoparticles and amplification products to achieve the signal amplification again. Combined with sensor microarray technology, the sensitive, rapid, high- throughput detection on six kinds of pathogenic microorganism by the biosensor chip specific can be realized, the detection method of biological sensor and traditional detection method can be evaluated by the methodology and statistics,and the quick thermostatic detection method of pathogenic microbe based on a solid rolling circle amplification phase nano- surface plasmon resonance biosensor can be created, provid ing a solid foundation for the identification and diagnosis of disease of pathogenic microorganism.Objective:To establish a nano-rolling circle amplification-surface plasmon resonance biosensor chip technology, which both has the direct contact with genomic DNA and signal amplification effect, providing a sensitive, specific, simple and rapid, high- throughput biological sensor method for the detection of pathogenic microbes.To study response mechanism and influence factors of surface plasmon resonance biosensor solid surface rolling circle amplification effect and prove the feasibility of surface plasmon resonance sensor amplification biological surface reaction signal, providing high sensitivity, high specificity detection solutions for complex samples of trace measured biomarker.Method:1. The design of the probe: Lookup 16 S r DNA genomic sequence of these six kinds of pathogenic bacteria in the Gen Bank database: Escherichia coli(E.coli), Enterococcus faecalis(E.faecalis), Shigella dysenteriae(S.dysenteriae), Streptococcus pneumoniae(S.pneumoniae), Staphylococcus aureus(S.epidermidis) and Staphylococcus aureus(S.aureus).Then screen target sequence for design of lock type probe, according to the target sequence design with lock type probe of Quick C utT M Hpa I enzyme cutting sites and the corresponding capture probes and universal sequence, at the same time, using Primer Premier 5 and O ligo 6 software, to predict the secondary structure of padlock probe, the capture probe, signal amplifying probe and universal sequence. The use of Array Designer software, maintains the consistency and good specificity between each probe.2. Establishment of liquid phase RCA amplification system: With six kinds of pathogens target sequence as a template, establish synthesis, liquid phase reaction of RC A amplification system, by agarose gel electrophoresis and real-time fluorescence quantitative PCR, verify the padlock probe, the capture probe and the template connection and amplification efficacy and specificity.3. Improvement of SPR biosensor: Based on previous research on the platform of SPR, the detection sensor and the biosensor chip surface self- assembly technology are optimized, sample flow rate control system, the detection channel and a the sensor temperature control device are improved.4. Biosensor chip surface membrane preparation and capture probe fixtion: O n the basis of biotin and streptavidin biotin covalent coupling, fix the capture probes with thiol modification immobilized on the surface of the chip, to explore the affect that capture probe nano gold particle values for SPR signal in the concentration that fixed on the chip surface probe.5. Optimization of RC A amplification system conditions of solid phase: Respectively discuss the connection efficiency and specificity, the optimal hybridization time lock type probe and target sequences at 40℃, 45℃, 50℃, 55℃, 60℃, 65℃ and 70℃ temperature conditions. The efficiency impact of 10 U, 15 U and 20 U E.coli DNA Ligase dosage on the probe connected into ring. The impact of the use of Exonuclease I and Exonuclease III on the specificity of the amplification system.To determine the optimal experimental conditions of solid RCA reaction of SPR biosensor chip surface.6. The solid phase reaction of RC A with nano gold signal amplification: Repeat signal gold nanoparticles modified RC A probes and amplification p roducts of solid surface for hybridization, which produce a stronger signal amplification effect. The experimental conditions of gold nanoparticle signal amplification system was optimized, and with synthetic DN A molecules as template to detect the sensitivity and specificity of the reaction system.7. Orthogonal test of multiple amplification detection: 6 pathogen specific capture probe are respectively fixed on 6 corresponding detecting pool, only to join with a pathogen template, repeated many times, the research method of cross validation can be multiple parallel amplification detection.8. Analysis of clinical samples detection and statistics: C linical specimens from 50 patients were assayed using SPR biosensor method of nano gold signal amplification, and were compared with the traditional pathogen detection methods(Dade Behring Micro Scan automatic microbial analysis system and the French bio Merieux ATB automatic microorganism identification method), and taken a Kappa test. Evaluate the established constant temperature type surface plasmon resonance biosensor detection method by methodology, to determine the specific steps for sample of clinical pathogenic bacteria detection.Results:1. Through the C lustal X2 and online BLAST comparison, six species of pathogenic bacteria(E.coli, E.faecalis, S.dysenteriae, S.pneumoniae, S.epidermidis, S.aureus) were found for the target sequence design locking type probe, according to the target sequence designed with a padlock probe Q uick C utT M Hpa I restriction enzyme site and corresponding capture probe, and the Primer Premier 5 and O ligo 6 software to predict the secondary structure of and capture probe padlock probe. Padlock probe connected into a ring has less secondary structure, with similar Tm values between the probes, so as to ensure the efficiency of RC A amplification and target sequence recognit ion specificity of the padlock probe.2. With six kinds of pathogens target sequence as a template, establish synthesis, liquid phase reaction of RC A amplification system, The RCA amplification products by agarose gel electrophoresis and fluorescence quantitative real- time PC R results showed that, with the target sequence pool has electrophoresis of RCA products with, no electrophoretic band in other lane. This shows that the lock type probe designed in this research can effectively and specificly identificated the target sequence of pathogenic bacteria, which can be used the detection of pathogenic bacteria in molecular level.3. Based on our preliminary research, solid-phase amplification signal amplification SPR sensor detector is optimized. SPR biosensor chip is gold plated film prism, gold film thickness is about 80nm; velocity control range is 5-1500μL /min, velocity control accuracy is ± 1 μL /min; Detection channel is 8 series flow pool; The temperature control range is 20℃-50℃, the precision is ± 0.1℃; The use of thiol end covalent modification, will assemble capture probe of gold nanoparticles to chip on gold film.5. At room temperature, biotin antibody immobilized on the gold film on the surface of the SPR sensor chip, Combined with nano gold streptavidin binding with biotin antibody covalently, finally respectively to six pathogens capture probes immobilized on the chip surface with six different flew in the pool and the best capture probe concentration is 1μM.6. Lock type probe and the target sequence of DN A hybridization temperature is 60 degrees centigrade, the efficiency of the cyclization of PLP connection, is the highest, the reaction specificity is the best; The best hybrid connection time is 30min; When E.coli DNA Ligase dosage is 10U、15U and 20 U, no significant difference in RCA products, so we chose 10 U as the best ligase dosage. When the padlock probe connected product does not use Exonuclease I and Exo nuclease III restriction, amplified nonspecific products appearance. The use of Exonuclease I and Exonuclease III restriction no-connected padlock probe, can improve the specificity of this detection.7. SPR biosensor detection method based on gold nanopar ticle signal amplification can improve detection sensitivity. In 10%(v/v) concentration, the signal of SPR diameter gold nanoparticles 15 nm value is relatively high. There were significant differences compared with the control group(P < 0.05). The limit of detection for synthetic template DN A molecule is 5 × 10-13 M((51.6 ± 7.2) × 10-5 RIU),and the concentration in the range of 5 × 10-13 M to 5 × 10-7 M is linear correlated(y = 503.5 x + 6126.0,R2 = 0.9853).8. Established SPR biosensor detection method based on gold nanoparticle signal amplification can complete the constant temperature on E.coli, E.faecalis, S.dysenteriae, S.pneumoniae, S.epidermidis and S.aureus for rapid detection in 3h, realizing multiple amplification detection for SPR biosensor chip surface of each stream pool. Various pathogens cross test showed that SPR angle signal value changes obviously(P < 0.01), which shows no cross reaction.9. Through the clinical specimens of 50 cases of SPR biosensor detection method of nano gold signal amplification, the detection limit of specimens of clinical pathogenic bacteria genomic DNA is 0.5 pg/μL((71.8 ± 8.1) × 10-5 RIU). Compared with the traditional clinical methods(Dade Behring Micro Scan automatic microbial analysis system and the French bio Merieux ATB automatic microorganism identification method), the sensitivity and specificity using this research method for the detection of clinical specimens are E.coli:92.6% and 95.7%;E.faecalis:100.0% and 100.0%;S.dysenteriae:100.0% and 100.0%;S.pneumoniae:94.7% and 93.5%;S.epidermidis:95.7% and 96.3%;S.aureus:100.0% and 97.3%. By Kappa test, results of the two detection methods have significant coherence(p <0.01, Kappa> 0.75).Conclusion:1. The padlock probe detection of six kinds of pathogenic bacteria E.coli, E.faecalis, S.dysenteriae, S.pneumoniae, S.epidermidis, S.aureus and the corresponding capture probe are successfully designed. The liquid phase and solid phase RC A amplification reaction system construction are optimized.This can identify target sequences in the background signal interference, also can achieve multiple connections and amplification reaction in a variety of target sequence. So as to realize Multiplex amplification reaction of the SPR sensor chip. Because of the experimental conditions are consistent, no cross reaction, which can be used for high-throughput detection for the biosensor.2. Thermostat Solid Phase RC A-SPR biosensor detection methods, which reduce the reliance on general gene diagnosis PC R amplification method. Because of their stable detection system, simple and convenient operation and the chip which can be reused, this facilitates the detection method for detecting SPR sensor applied in clinical specimens.3. Using level signal amplification of the avidin biotin syste m, solid phase reaction of RCA signal of the two stage amplifier and the signal of gold nanoparticles three stage amplifier, effectively improve the response signal of SPR biosensor chip surface value, which can significantly improve the sensitivity of SPR biosensor. The method for the rapid detection of trace molecules SPR sensor provides a reliable detection platform. The method will be more and more used in aspect of rapid detection of nucleic acid at the molecular level.4. Method for detection of thermostatic type solid phase RC A-SPR biosensor, which can not only ensure the specificity and sensitivity of detection in pathogenic bacteria, but also can quickly realized RC A solid phase reaction in real time and on- line unmarked monitoring,which provides a new method for nucleic acid amplification and identification of the molecular level.5. Thermostat type SPR sensor detection method which was successfully constructed can detect the six parallel common clinical pathogenic microorganisms within 3 hours. Detection method is fast and easy, no mark, samples need less, rapid, sensitive and specific characteristics. Compared with the traditional clinical methods and gene chip technology, this reduce the technical requirement and test cost.This provides a new ide a for the rapid detection of common clinical pathogenic microorganism and other trace gene diagnosis, to solve the problems existing in the clinical routine detection method, to meet the clinical rapid, accurate diagnosis demand. Its application prospects will be very broad. |
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