| BackgroundBacteremia-induced severe sepsis is the leading cause of morbidity and mortality worldwide, particularly in patients who are immunocompromised. In last decades, various techniques have been developed for the clinical diagnosis of bloodstream infections, such as conventional blood culture techniques, enzyme-linked immunosorbent assay, immunoassay, sequence analysis, and so on. These conventional methods are generally reliable but time-consuming, laborious and expensive. Therefore, it is desirable to explore some simple, sensitive, and low-cost diagnostic methods to detect pathogenic bacteria in clinical blood samples. 16S rRNA sequence analysis has been used to clarify the taxonomic affinities of a wide range of taxa and as a powerful tool for the detection of pathogenic bacteria.Because of sensitivity, simplicity and cost-effectiveness, piezoelectric quartz crystal microbalance (QCM) biosensors have showed attractive roles in various molecular analysis techniques Recent years, piezoelectric biosensor is becoming a focus in biomolecule detection field. It could detect many biomolecular real-timely, sensitively and specifically, such as DNA, antibody, enzyme, bacterium, based on different recognition elements. In recently research, it had been used in many research fields such as molecular recognition, clinical diagnosis, molecular filming, surface property research, poison research and so forth. However, for most of the previously reported nucleic acid biosensors for bacterial detection, their sensitivity and detection limit of are still difficult to meet the demand of clinical analysis. In this study, a QCM DNA biosensor array was developed to rapidly detect 5 common pathogenic bacteria which are the major causes of bloodstream infections. Moreover, in order to improve the detection sensitivity of QCM nucleic acid biosensors, two signal amplification strategies have been developed, which includ enzyme and nanoparticles signal amplification. The sensitivity and specificity of the QCM system were evaluated. The application of the QCM system was tested in real clinical blood samples. This study lays the groundwork for applying this QCM DNA biosensor array in the labs and for rapid detection of pathogenic bacteria.Methods1. The QCM DNA biosensor array was constructed which includes electronic oscillation circuit, voltage stabilizer, thermal controller system, and 2×5 detection wells. The new software PESA 4.0 was designed based on Visual C++, which was interfaced with the QCM detector. The stability of the new system was tested in gas phase and liquid phase individually. The deoxidized thiol-modified probe immobilization was developed,and the experimental conditions of the immobilization methodwere optimized.2. The universial primers were designed to be complementary to the conserved regions of bacteria 16S rRNA gene using Primer Premier 5.0 software. The specific oligonucleotide probes were designed to be complementary to the highly variable regions of 16S rRNA gene in target bacteria by Array Designer 4.0, which include Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis, Escherichia coli. The experimental conditions for the biosensoring system were optimized.3. Enzyme and nanoparticles signal amplification strategies were developed. The sensitivity and specificity of the current QCM biosensor system based on these two signal amplification methods were tested. In addition, Faradaic impedance spectra measurements were carried out to demonstrate the alteration of the interfacial properties of the QCM electrodes during the signal amplification process.4. To demonstrate the use of the biosensing system for the identification of pathogenic bacteria, 108 blood culture samples were detected individually using the QCM nucleic acid array and the conventional microbiological techniques. Chelex 100 and alkali lysis method were investigated. The clinical sensitivity and specificity of the current QCM biosensor array system for bacteria detections of clinical samples were also tested.Results1. The size of the new biosensor array has been minimized to (25.0±1.0)×(35.0±1.0)×(15.0±1.0)cm(L×W×H).Moreover, the QCM biosensor array has strong vibration ability and table frequency in liquid phase. In ten minutes, the changes of frequency were less than±1Hz both in gas phase and liquid phase. The deoxidization of thiol-modified probes significantly increased immobilization efficiency (p<0.05) and we selected the probe concentration of 2.0μM as the optimal immobilization concentrations for the following experiments.2. The experiments showed these target bacterial could be successfully amplified by one-time PCR with the common primers. The results also showed that the premiers were in good repetitivety. The designed probes have strong specificity and great reliability. In addition, the difference of Tm value between the probes was only 0.5℃, hence they had the almost same hybridization conditions and could meet the requirements of the biosensor array detections. The optimal hybridization temperature is 40℃experiments and the optimal hybridization time is 90 min.3. The use of both Au nanoparticles and enzyme effectively amplified the signals in frequency shifts due to the relatively large mass compared to DNA targets, and resulted in a improved detection limit of 2.0×103 CFU/ml and 2.0×102 CFU/ml, respectively. The frequency shift caused by the Au nanoparticles method(271.6±12.1Hz) was significantly larger than that caused by enzyme method (127.5±6.5Hz) (P<0.01). Therefore, enzyme method was used in subsequent experiments.4. The alkali lysis method was the most sensitive, reproducible, simple and cost-effective extraction method to extract bacteria DNA in clinical blood cultures. Compared with conventional microbiological method, the diagnostic sensitivity of the assays resulted in 90.2 % and the specificity resulted in 98.5 %. This test system allows identification of 5 bacteria within ca. 4 h.Conclusions 1. The developed QCM DNA biosensor array has strong vibration ability and table frequency in liquid phase, and it is simple and cost-effectiveness. The new QCM DNA biosensor system can provide a reliable plateform for gene detection.2. The designed universial primers and specific oligonucleotides can increase the detection flux of the current QCM DNA biosensor array system and simplify the process of the identification of pathogenic bacteria.3. Both enzyme signal amplification and Au nanoparticles amplification system could detect target DNA specifically and amplify the piezoelectric signal significantly. It has great versatility and can be used in different types of piezoelectric biosensors in detecting tiny mass biomolecular.4. For clinical blood sample detection, the developed QCM DNA biosensor array has good clinical sensitivity and specificity compared with the conventional microbiological method, which suggest that this QCM nucleic acid biosensor array with enzyme amplification method can be expected to be a potential clinical diagnosis method for rapid detection of pathogenic bacteria causing bloodstream infections or other microorganisms in clinical samples.
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