Study On Molecular Biology And Novel Diagnosis Techniques Of Lyme Disease

Lyme disease is a kind of rock-ribbed infectious disease caused by the tick-borne spirochete Borrelia burgdorferi, which is at the bottom of multisystem disorder disease and was described as a new entity in the United States in the late 1970s. Lyme disease is distributed broadly in the world and has been reported in more than 70 countries in 5 continents so far. The endemic areas of the Lyme disease are still enlarging, incidence of it is increasing every year and the disease has become a severe health problem to human beings. So, WHO has ranked it a new infectious disease, which needs focus study and prevent. In the United States, the Centers for Disease Control and Prevention (CDC) initiated surveillance for LB in 1982, and the Council of State and Territorial Epidemiologists adopted a resolution making LB a nationally notifiable disease in 1990. In China, Lyme disease occurred in the forest areas of at least 29 provinces. Pathogen evidences showed that there are natural endemic areas of Lyme disease in the forest areas of 19 provinces. In the current 30 years, there are 40 species emerging infectious diseases, it means that should be emerging one infectious disease every year. 15 EIDs including LB are epidemic in China. It is very important to study Molecular Biology of LB today, it is the base of study on pathogenesis,diagnostic technique and vaccine.Multiple clinical manifestations of Lyme disease were easily confused with other disease. Infection with Borrelia burgdorferi can result in dermatological, neurological, cardiac, and musculoskeletal disorders. So the laboratory diagnosis is significant. Several methods have been used for detection of antibodies to Borrelia burgdorferi. They included indirect immunofluorescent-anti body assays (IFA), enzyme-linked immunosorbent assay (ELISA) and the western blot (WB). Each of them has its advantages and disadvantages. The journal of Nature Genetis publish an investigative report, it lists 10 kinds of pivotal biologic techniques which are the most potential to improve the healthiness of the people all over the world especially in developing country. The first one is molecular diagnostic technique of infectious diseases. About half death cases are arose by infectious diseases, and almost diagnostic techniques are expensive and difficult to operate.The new technique of Point of Care Testing (POCT) is very simple and fast to operate. The experts forecast that the POCT will reach to 80% and change current status in 5 years. Because the quantum dots (QDs) have unsurpassable character to organic fluorescent dye, it is lighting undoubtedly to develop the technique based on QDsFIA and POCT.The most important characteristics of SPR sensor are its versatility and capability in real time monitoring the association or dissociation of biomolecules on the surface of the sensor without the need for fluorescence or labeling of the biomolecules. These characteristics made the SPR technique an easy, convenient and reliable one for determining the concentration and molecular weight, monitoring change in structure, measuring kinetic constant and binding specificity of individual biomolecules. For above reasons the SPR sensors grow as a new powerful technology in chemical and biological field. Since then, the research and application of SPR technology have shown extensive growth and gradually become the hot spot and research frontier in the biosensor field in the world. So SPR is befit to diagnose LB and distinguish the genotype of Borrelia burgdorferi.In this paper, we collected the ticks, which tested to be Ixodes Persulcatus in Mount Changbai, Fusong County, Tonghua County and Changbai County .The seven Borrelias have been isolated from these Ixode ticks, the dominant species of tick in north China. All Borrelia isolates were grown in BSK medium at 33°C. Extraction of DNA was performed as described previously. PCR technique was used to amplify the OSPA gene from the whole cellular DNA of isolate of Borrelia burgdorferi. The results show that the ticks that we colleted were tested to be Ixodes Persulcatus. The rate of the ticks with Borrelia is 38%. The OSPA genes of the isolates were 742bp. Jilin province is one of natural endemic areas of Lyme disease.The novel LB diagnosis sensor and technique based on SPR was developed firstly. The interactions between Borrelia burgdorferi and antiserum were presented. The binding rate of antibiotics and protein was studied by this apparatus. According to these differences the genotype of Borrelia burgdorferi (B.afzelii and B.garinii are familiar in Jilin area) can be distinguished. The method was shown to have many advantages over traditional methods. The major advantages include the elimination of interference of across reaction, high sensitivity and screening rapidly. The potency excelled to 1:6400, the cut-off-point for diagnosis Lyme disease is defined by the result of 20 positive samples and 40 negative samples, the measurement result of 12 sample groups and 40 control groups and the evaluation with version 16.0 SPSS software have obtained good results.The novel LB diagnosis technique based on QDsFIA was developed firstly. Organic fluorophores limitations include narrow excitation bands and broad emission bands with red spectral tails, which make the simultaneous evaluation of several light-emitting probes difficult due to spectral overlap; Quantum dots (QDs), also known as semiconductor nanocrystals, have recently emerged as promising alternatives to organic dyes in IFA applications. Firstly, their emission spectra are narrow, symmetrical, and tunable according to their size and material composition, allowing closer spacing of different probes with less spectral overlap. Secondly, they exhibit excellent photostability and broad absorption spectra which make it possible to excite all QDs simultaneously with a single light source and minimize sample auto-fluorescence by choosing an appropriate excitation wavelength. Thirdly, they show higher luminescence and quantum yield than conventional fluorophores under appropriate conditions. They are strongly luminescent and their emission wavelength can be tuned between blue and red wavelengths by changing the particle size. The method major advantages include the elimination of interference of across reaction because of Fluorescence resonance energy transfer (FRET), high sensitivity and screening rapidly. The potency excelled to 1:6400, the cut-off-point for diagnosis Lyme disease is defined by the result of 20 positive samples and 40 negative samples, the measurement result of 12 sample groups and 40 control groups and the evaluation with version 16.0 SPSS software have obtained good results.The novel the genotype of Borrelia burgdorferi identification technique based on SPR was developed firstly. The interactions between Borrelia burgdorferi and antiserum were presented. The binding rate of antibiotics and protein was studied by this apparatus. According to these differences the genotype of Borrelia burgdorferi (B.afzelii and B.garinii are familiar in Jilin area) can be distinguished. One determinate case with clinic evidence confirms the technique serviceable.