Detailed Study On The Development Of Magnetic Separation And Chemiluminescence Based Biosensor For The Simultaneous Detection Of Multiple Viruses

Development of Magnetic Beads based protocol for the purification of high quality nucleic acids from cell, bacteria and virusThe isolation of nucleic acids (NA) is the preliminary step to carry out genetic studies and DNA biosensor development. The presence of inhibitors in the purified NA interferes with the downstream application. These salts and other organic contaminations particularly challenge the analytical sensitivity of DNA biosensors. We proposed magnetic beads based NA purification protocol. The detailed study was carried out to optimize the factors which might affect nucleic acid purification. The results suggested that 6 M guanidium hydrochloride salt concentration was critical for NA isolation. The inverse relation has been found in the pH of the lysis buffer and quality and quantity of NA. The NA yield was relatively stable at pH 4-5. It has been observed that the use of carrier RNA was indispensable for viral genome isolation. The addition of ethanol to lysate in 1:1 ratio greatly improved NA recovery. The elution efficiency of DNase and RNase free water, 1X TE buffer and 2X PCR buffer was compared. The carrier RNA was best eluted in DNase and RNase free water and 1X TE buffer. The larger elution buffer’s volume showed positive effect when NA was recovered from large number of cells. A simple method was established to extract NA from cells, bacteria and serum. The purified NA was successfully amplified in PCR and RT-PCR to verify the reliability of established protocol.Highly sensitive detection of HBV based on PCR amplification and Chemiluminescent DetectionHBV infection has been considered to develop a number of serious diseases such hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma and non-Hodgkin lymphoma. The early diagnosis of this deadly infection is vital to start treatment and restrain disease spread. The current molecular detection systems are expensive and require highly skilled labor to perform this test and interpret data. As an alternative, a highly sensitive and automatable method for HBV detection from clinical samples by combining PCR, magnetic separation and chemiluminescent technique has been developed. Annealing temperature of the primers, Polymerase chain reaction (PCR) buffer concentration, magnesium chloride concentration (MgC22), primer concentration and amount of Taq DNA polymerase were optimized to obtain maximum product yield. The length of probe sequence, the position of the probe and GC contents of the probe were found to be very important parameters for producing high chemiluminescent signal. Probe-template hybridization temperature was optimized to be 45 ℃. The optimized probe concentration was 5 μM. Optimum hybridization time was determined to be 30 minutes.50 μg of probe modified MBs were found to be balanced amount of MBs for generating maximum chemiluminescent signal. Negative signal was obtained when specificity was checked by replacing HBV with HCV and HIV infected serum. This method displayed excellent sensitivity by detecting 10 viral IU/ml of serum. Considering that 100 μl of serum was initially used for HBV DNA extraction, it can be claimed that the developed system detected 1 IU of HBV per reaction.Magnetic separation and Chemiluminescent based highly sensitive method for HCV detectionHepatitis C virus (HCV) is one of the major threats in the world. Infection with this virus leads to the development of chronic hepatitis, cirrhosis and primary hepatocellular carcinoma (HCC). Developments of cheap diagnostic tools for early detection of HCV are highly desirable in developing countries. In this report, chemiluminescent based genoassay for highly efficient HCV detection has been described. Amplification of HCV RNA was carried out in one step reverse transcription-polymerase chain reaction (RT-PCR) to achieve high sensitivity. Annealing temperature of the primers was determined to be 58 ℃. PCR buffer was used in 1.5 X concentration for optimum performance. The concentration of MgCl2 and primers were optimized to be 2.4 μM and 200 nM respectively. The optimized amount of hot start Taq DNA polymerase was 1.5 U/50 μl reaction. We found that probe length, position and GC contents greatly affect detection signal. Hybridization of probe and template occurred best at 55 ℃. The optimum probe concentration was 5 μM and maximum chemiluminescent signal was recorded when hybridization was allowed for 30 minutes. The assay successfully detected 10 viral IU/ml of serum with excellent specificity. In fact, the assay detected 1 IU/reaction as 100 μl serum was initially used to extract HCV genome.Magnetic separation and Chemiluminescent based highly sensitive method for HIV detectionHuman immunodeficiency virus (HIV) is a lethal virus and causal agent of Acquired immune deficiency syndrome (AIDS). More than 25 million deaths have been caused by HIV since its discovery.2.7 million new HIV infections are reported every year. It is important to explore new cheap and effective means of viral diagnosis. This report describes the HIV detection assay based on DNA hybridization. The detection was finally realized by using chemiluminescence. One step reverse transcription polymerase chain reaction (RT-PCR) was used to amplify HIV target sequence.58 ℃ was used as annealing temperature. The biotin-11-dUTP was incorporated into target sequence during RT-PCR amplification. Probe modified MBs were used to capture these target sequences and generate detection signal. Different factors which might affect bioconjugation event were optimized. The hybridization temperature was 35 ℃.5 μM probe concentration produced maximum chemiluminescent signal. The optimum hybridization time was 30 minutes. This method shows excellent specificity. Negative signal was obtained when HCV and HBV infected serum were used. This system successfully detected 10 viral particles/ml of plasma.Development of a chemiluminescence based assay for simultaneous extraction and detection of multiple virusesNucleic acid testing (NAT) based methods are more sensitive, specific and preferred over enzyme immunoassays. Assays have been designed to detect multiple pathogens in order to reduce the high cost of NAT. However these assays can’t reliably detect large number of pathogens. In this report, a DNA hybridization based chemiluminescent detection method has been proposed for stable multiplex detection without any limit on the number of pathogens. The idea was practically demonstrated by carrying out simultaneous extraction and detection of HBV, HCV and HIV. The probes showed higher specificity by capturing only targeted viral sequence. When the sensitivity of each virus was detected in the presence of higher load of other viruses, the assay could detect 10 HBV IU,10 HCV IU and 100 HIV IU per ml of plasma. This assay can be easily modified into an automated system for high throughput detection because of the use of magnetic separation technology from nucleic acid extraction to the chemiluminescent detection.Semi-Automated system for multiplex viral detectionClinical labs are the front line fighters against any infectious disease. They play a very important role in infection control. New threats are emerging day by day for example HIV, Anthrax, Ebola etc. It emphasizes the need for the development of automated systems for high through put screening and diagnosis of threats. We have developed magnetic beads based NA extraction protocol and we have successfully used it for the development of highly efficient biosensor for the multiplex detection. We demonstrate that this method can be automatized for high through put detection. A simple experiment was conducted to optimize the different parameters of automated NA extractor to simultaneously extract HBV DNA and HCV RNA. We find out that holding of magnetic particles by magnetic arm during the washing Ⅰ and washing Ⅱ is very critical. The downstream RT-PCR failed to produce any bands, when magnetic arm released NA bound MPs in wash Ⅰ and wash Ⅱ for thorough washing. RT-PCR produced sharp desired bands when opposite conditions were provided by holding the MPs tightly by magnetic arm of the automated extractor. Other factors like movement of magnetic arm, for mixing the reaction components, were also studied. The optimized protocol was successfully employed to extract HBV and HCV nucleic acids. In short, a semi-automated system for simultaneous detection of HBV and HCV has been demonstrated.