Development Of An Noval Biosensor For The Detection Of Viruses Based On Magnetic Nanoparticles And Chemiluminescence

1. Development of Magnetic Beads based protocol for the purification of high quality nucleic acids from cells, bacteria and virusThe extraction of nucleic acid is recognized as one of the most essential steps in molecular biology for initiating other downstream applications such as sequencing, amplification, hybridization, and cloning. Recent advances in the development of nucleic acid extraction systems have increased rapidly, among which magnetic nanoparticles (MNPs) have received great attention and significant interests due to its facile manipulation and low cost. Besides, extraction techniques using MNPs have great benefits because of its shorter processing time, ease for automation and limited use of chemicals.In this study, a method for simultaneous extraction of DNA and RNA from bacteria, cells and serum based on magnetic nanoparticles (MNPs) was described. Lysis buffers were prepared to help the nucleic acid released and adsorbed to MNPs. Then, two washing buffers were used to remove the contamination of proteins and carbohydrates. The nucleic acids were finally eluted by Deoxyribonuclease (DNase) and Ribonucleases (RNase) free water. Different factors which might affect the purification of the nucleic acid were investigated, and the quantity and quality parameters of the nucleic acid were also recorded. The DNA and RNA extracted from bacteria were then respectively subjected to polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) to further confirm its quality. The results indicated that our method can be successfully used to simultaneously extract DNA and RNA from biological samples.2. Highly sensitive detection of HBV and HCV based on multiplex PCR and Chemiluminescent DetectionMethods based on nucleic acid testing (NAT) are more sensitive, specific and preferred over enzyme immunoassays. Recently, some bioassays have been designed to detect multiple pathogens in order to reduce the high cost of NAT. However, those assays cannot reliably detect large number of pathogens. In this study, a DNA hybridization based chemiluminescent detection method has been investigated for stable multiplex detection without any limit on the number of pathogens. The idea was practically demonstrated by carrying out simultaneous extraction and amplification of Hepatitis B virus (HBV) and Hepatitis C virus (HCV) through one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). The detailed study was conducted to optimize the factors which could affect chemiluminescent signal. The results showed that the probes for HBV, HCV and HIV possessed higher specificity by capturing the target sequence.10 viral copies/mL of serum were detected in monoplex detection protocol for HBV and HCV. However, when the sensitivity of each virus was analyzed in the presence of higher load of other viruses in the multiplex detection assay, the assay could finally detect 10 HBV copies,10 HCV copies and 100 HTV copies per ml of plasma. Furthermore, magnetic separation technique has been used throughout the experiment, from nucleic acid extraction to the chemiluminescence detection, which enabled this system to be modified into an automated platform for high throughput applications.3. Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and ChemiluminescenceRNA viruses, particularly, the highly pathogenic avian influenza (HPAI) virus, pose serious health concerns, and cause huge economic losses worldwide. Diagnostic tools for the early detection of these deadly RNA viruses are urgently needed for implement treatment and disease control strategies. Conventional reverse transcription polymerase chain reaction (RT-PCR)-based chemiluminescent (RT-PCR-CL) detection is frequently used for the diagnosis of viral infections. However, the requirements for expensive PCR machines and longer thermocycling times are serious drawbacks. In this study, we propose a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with chemiluminescence (CL) to detect H7N9 virus. The proposed method does not require any expensive instruments, and processing time is remarkably shortened compared with that of RT-PCR-CL. Since several factors including RT-LAMP temperature, probe concentration, hybridization temperature, and hybridization duration might affect the CL signal, each of these parameters was investigated and optimized. One thousand copies/mL of H7N9 RNA were detectable using the optimized RT-LAMP-CL method. The detection time was significantly reduced by using RT-LAMP, in comparison with conventional RT-PCR-CL. This technique holds great promise for viral detection and diagnosis, especially with regard to avian influenza virus.