Research Of Clostridium Perfringens Piezoelectric Biosensor Gene-chip Detection System

Objective1. To construct hardware and software for QCM system for detecting clostridium perfringens. And design three kind of gene-chips for three types of C. perfringens toxin, which are C. perf ringensαtoxin, C. perf ringensβtoxin and C. perf ringens enterotoxin.2. To search a method for amplifing C. perfringens and their three toxins'gene by multiplex PCR. Using the QCM system to detect DNA sequence amplified by multiplex PCR, and optimized the QCM detecting conditions.Methods1. On the foundation built on previously researching, we construct the detection system,including its construction and electrical circuit for QCM. With the fine processing technology, we make up of the sensor of QCM and adjustable pluck-plugged 2×5 style gene sensor array. For raise of the adaptivity of QCM system, we apply a new frequency count card and a new transmission mode with USB. We also add a temperature control unit to fit the demand of biology response. For improving human-machine interface and adding new function , we develop the new software for recording and analysing the changing frequence of QCM, called QCM-biosensor PESA-4.0, based on the PESA-2.0 developed earily.2. Fine processing technique was used to make single quartz crystal wafer, AT cutting with fundamental frequency 10MHz. And then, the gold electrode around two sides of the sensor was plated by the technique of ion sputtering. On the one side of gold electrode, we immobilize oligonucleotide probe to construct gene-chip with thiol-self-assembly technique. The oligonucleotide probe are designed by myself ,using nucleic acid analyse and design software, Array Designer V2.0,Fast PCR V3.6.5, Primer Premier V5.0, Oligonucleotide Properties Calculator. They are three types, base on three sequence of C. perfringens toxin genome, which are C. perf ringensαtoxin gene, C. perf ringensβtoxin gene and C. perf ringens enterotoxin gene. 3. Using two methods for extraction of C. perfringens'genome, including kit extraction and boiling extraction after incubating with enzyme. Searching a method to amplify C. perfringens toxin by multiplex PCR. The mutiplex PCR was developed with three sets of primers designed on the sequences of three Clostridium Perfringens toxin genes( CPα,CPβand CPE ) published in GeneBank, with the help of nucleic acid analyse and design software, which are list above. At last, amplifying a simulated sample and a mixed sample by the designed system of multiplex PCR to test the reliability of reaction system.4. Detecting the target DNA sequence obtained at step3 by QCM system. At the same time, we research the best reaction conditions, including pH value, ion intensity and temperature. At last, we detect the C. perfringens'simulate-sample with the optinal reaction condition by QCM.Results1. The C. perfringens QCM system is developed by ourselves. Detecting temperature can be controlled in the range of 20~70℃by temperature control unit. It takes less than 45min to raise temperature form 20℃to 70℃in the unit, and precision is±0.5℃. The system works stably. In air phase, the frequency fluctuates in 2Hz, drifting±5Hz in 2 hours. In liquid phase, the frequency fluctuates in 5Hz, drifting±10Hz in 2 hours respectively. All system is reliable and easy to operate, fitting to detect gene.2. The software, PESA-4.0, works well. It runs in Microsoft Windows Operation System with good human-machine interface. All of its parts show clearly in windows, read easily and directly. The result shows that the programme can be fit for detection.3. Three expected sequences were obtained successfully by multiplex PCR and identified by electrophoresis. The consistency of PCR amplification results is quite good with two different extraction methods. Obviously, the method of boiling extraction after incubating with enzyme is better because it is convenient and cheap.4. The best reaction condition is pH7.6, 0.64mol/L Na+ and 37℃in the QCM system. Under the best reaction condition, the detecting results show that the frequency decreases obviously. And the value of decreasing frequency is corresponding to with the designed sequence length, detected by three types of genechip. Conclusions1. The QCM system works reliably.2. The three specific sequences of Clostridium perfringens toxins could be amplified by multiplex PCR system.3. The method of boiling extraction after incubating with enzyme is feasible with advantage of convenient and cheap.4. The system can be used to detect C. perfringen and genotyping their toxin.