Rapid Detection Of Zoonotic Pathogens Using Leaky Surface Acoustic Biosensor And Rolling Circle Amplification

Objectives:1. To build a novel Down Plug-in Leaky Surface Acoustic Wave (DP-LSAW) biosensor system which be more suitable for analyzing biomolecules in liquid for increasing the stability and accuracy of biosensor and high-flux detecting several samples simultaneously.2. To rapidly detect several zoonotic pathogens using the novel LSAW biosensor for the earlier infection diagnosis.3. To establish a convenient, rapid and low cost rolling circle amplification (RCA) technique for the detection of single base mutations of influenza A H1N1 M2 and NA genes.Methods:Fabrication of Down Plug-in LSAW biosensor system and detection of relative zoonotic pathogens1. A new Down Plug-in LSAW biosensor, consisting of twin LSAW delay lines channels fabricated on 36? rotated Y-cut X-propagation LiTaO3 piezoelectric single crystals , was developed according to the construction of previous LSAW biosensor . Input IDT, out put IDT and their lead wires were coated with a thin SiO2 layer to protect them from water. Piezoelectric substrate thickness and size of the sensor were reduced so that it could be inverted vertically into the reaction pool.2. Fixed size reaction pools were made of medical polypropylene materials.3. 2×3 array of biosensors were designed.4. The temperature control equipment based on the principle of semiconductor cooling chip was composed of power source, temperature control panel and heating or cooling system components.5. The down-plug-in LSAW biosensor detection system was fabricated of DP-LSAW biosensors, NI-PXI signal genesia and acquisition system, temperature control equipment, computer and LabVIEW software .6. Specific capture probes were designed for each pathogens including rabies virus, foot-and-mouth disease virus, Bacillus anthracis and Streptococcus suis. The hybridization between target DNA sequences and capture probes was detected real time by DP-LSAW biosensor.Detection of single base mutations of influenza A H1N1 M2 and NA gene using RCA1. M2 and NA gene of H1N1 were target genes in this research. Two circularizable probes were designed to detect these genes. Circularizable probes were ligated to closed rings by DNA ligase when they specifically bond to target genes which had single base mutation. After the rolling circle amplification, the single base mutations were specifically detected.2. Optimal conditions for RCA method were studied, and the specificity and sensitivity of the method were determined by testing wild and mutation type artificial templates.3. The specimens from clinic were detected and compared with sequencing method to confine its accuracy.Results:1. DP-LSAW biosensor detection system was successfully established .The instrument was small and its appearance was exquisite. Biosensor chips could be fixed and unloaded conveniently. The center frequency of biosensor was 100MHz , and the range of operating temperature was (20~70)℃, accuracy of±0.5℃.2. DP-LSAW biosensor detection system could directly detect the hybridization between capture DNA probes and target DNA of pathogens real time. The outcome signal was very stable when biosensor worked in liquid, the acoustical noise is 0.005 degree. The output phase changes were more than 0.01 degrees when ng/μl target DNA were detected by 1μm/L capture DNA probes. The signal to noise ratio was over 2 degrees. The reaction time was between 15min and 25min.3. Adopting heat cycle ligation and increasing the ligation temperature(65℃)could make sure the specificity of RCA. Through series detection of the target sequences with different concentrations, the lowest level of mutations detected by RCA was 1% of the total. RCA detection results were consistent with the sequencing method.Conclusions1. DP-LSAW biosensor detection system could directly detect the nucleinic acid hybridization reaction of zoonotic pathogens real time. The stability and specificity of biosensor was good, the time of test procedure was short, but the sensitivity of DP-LSAW biosensor was still need to be increased.2. RCA technique was successfully built to detect single base mutation of influenza A H1N1 M2 and NA genes. The sensitivity and specificity of this method met the testing requirement.