Experimental Studies On DNA Biosensors For Rapid Detection Of Pathogenic Microorganisms

The DNA recognition technology is continuously improved with the development of molecular biology and biotechnology.Compared to traditional detection methods based on phenotypic characteristics of the pathogenic microorganisms,the molecular diagnostic techniques based on DNA recognition technology are the preferable choice for the detection and identification of the pathogenic microorganism since they can offer the high speed,sensitivity and specificity.The information obtained from genome sequencing projects has opened the door to tremendous biological analysis and detection.DNA sensor technology integrates molecular biology with physics,chemistry and micro-electronic technology,has advantages of high speed,sensitivity and specificity,and high throughput. It can be used in the fields of biological engineering,clinical medicine, environmental monitoring,foodstuff analysis,and so on.According to label or not,the DNA biosensors can be divided into two categories,one is label-based DNA biosensor and another is label-free biosensor.The label-based DNA biosensor is based on changes of optical and electrochemical signal caused by signaling molecule.The label-free biosensor is based on changes of optical,mass or electrochemical signal caused by hybridization of nucleic acid.The dissertation includes two parts.In partⅠ,employing the ribosomal gene-typing technology,DNA biosensor technology and nano-amplification technique,a label-based DNA biosensor technique for simultaneously detection and identification clinical common pathogenic fungi was developed.Due to the high efficiency,low cost,good specificity and sensitivity,this technique offers a reliable alternative to conventional methods for the detection and identification of clinical-important fungal pathogens.In partⅡ,we explore a label-free biosensor based on surface plasmon resonance(SPR) and dimeric peptide nucleic acid(bis-PNA) as a platform for detection of indicator free DNA.The main results were as follows:PartⅠ:A DNA biosensor based on nanogold for simultaneously detection and identification clinical common pathogenic fungi.1.In accordance with the literature report and the clinical investigation,20 kinds of clinical important pathogenic fungi were selected as the target pathogen in this study,including Candida, Aspergillus,mucoraceae,dermatophytes,and so on.2.Based on the multi-alignment of the ribosome RNA(rRNA) gene sequences among 30 species of pathogenic fungi belonging to 14 genera,a pair of universal primers were designed for the amplification of the ITS2 regions of rRNA.The forward primer is complementary to the conserved sequence of 5.8S rRNA,and the reverse primer is complementary to the conserved sequence of 28S rDNA.A universal PCR amplification method was established and optimized to common pathogenic fungal strains. Using this PCR amplification method,PCR products of 20 target fungi showed target band of 259-531 bp respectively,and all non-fungal samples were no PCR products.3.By using GeneReleaser,the procedure of the preparation of DNA templates is much more simple and faster(0.5h) than that of conventional methods(＞2h).4.The species-specific oligonucleotide probes targeting ITS2 sequences were designed using AlleleID 6.0,analyzed and screened by bioinformatical technology,then verified by a universal PCR combined reverse blot hybridization.It turned out that the selected probes were all of high specificity and reliability,and had the same hybridization conditions.5.A DNA biosensor technique integrating nano-gold labeling,silver stain enhancement and the microarray technique was developed and optimized,including the optimal spotting concentrations of probes,the optimal hybridization temperature and time,the optimal procedure of silver stain enhancement and perfect self quality control system.The results showed that the method had ideal specificity and its detection sensitivity reached 50fmol/L for amplicon of Candida tropicalis and 10 cells/ml for Candida albicans.The hybridization signal can be observed visually or recorded by optic scanner.6.The method was further verified by fugus cultures from clinical specimens,mimic clinical samples spiked with standard fungal strains and clinical infectious samples.It was showed that the results of the DNA biosensor were completely consistent with that of traditional techniques, and our method was feasible for detection of clinical samples obtained from patients suffering from fungal pathogens.Therefore,the experiments mentioned above have proved that established nanogold-amplification technique-based DNA biosensor combining with universal PCR provides a simple,rapid,sensitive and specific method for the identification of fungal pathogens.The analysis is simple to perform and can be completed in 6 hours,compared with 2～4 days for conventional methods.Compared with the classical DNA biosensor,our method do not requires the expensive labeling material and complex detection equipment,so the efficacy/cost is greatly increased.It can meet the high throughput demand of clinical detection,and promise to be a alternative to conventional methods for the detection and identification of fungal pathogens in the clinical laboratories.PartⅡA label-based DNA biosensor based on SPR and bis-PNA 1.In this study,a parallel scan SPR imaging technique was adopted into SPR biosensor.This technique has the advamage of high sensitivity as the other SPR technique.The technique can offer two-dimensional quantificational distribution map of reflective index(RI) on the sensing plane.The pixels on the map have brightness linearly correspond to their RI.Another advantage of this technique is that the parallel scan method makes it a fast imaging,high-throughput analyzing technique.2.A specific bis-PNA probe was designed for M gene of influenza virus.The cytosine bases in N-terminal half of bis-PNA are replaced by pseudoisocytosine bases(J base),which do not require protonation to form triplexes,so it has high affinity to dsDNA and high specificity at neutral pH.3.The thiol-modified bis-PNA probe was immobilized to the surface of gold membrane by Au-SH bond.The dsDNA could hybridize with bis-PNA without denaturation.The detection sensitivity of the method reached 1pmol/L.Combining with SPR senseor based on parallel scan SPR imaging technique and bis-PNA probe,a label-free DNA biosensor was built and the preliminary verification tests were conducted.According to the result, the target dsDNA can hybridize with bis-PNA and be directly detected without any label and denaturation.The biosensor technique provides a simple,rapid,and high throughput means for the detection of nucleic acid. However,for application in clinical diagnosis,some conditions of this technique should be further optimized and improved,such as the probe design,hybridization conditions,the stability of detection systems,etc.