Pilot Study Of MLPA Diagnostic Kit For The Pathogens Of Central Nervous System Infection

Central nervous system(CNS)infection is a common clinical disease, due to high mortality and severe sequelae it caught great attention. Rapid clinical diagnosis is a crucial for better treatment of CNS infections. But there is no quick and accurate diagnostics presently.We first summarized the common CNS infection pathogen species and designed the MLPA diagnostic probes for 17 common bacterial pathogenes of intracranial infection. The key reagents to construct MLPA probe plasmids were patented by the MRC-Holland company. In addition to the preparation of short probes using synthetic method and we had to establish a set of method for generating the long MLPA probes step by step. We used the M13 phage plasmid to prepare 17 bacterical species of long MLPA probes. Every M13 phage vectors was verified by sequencing to ensure that all of 17 vectors could be used for the preparation of the long MLPA diagnostic probes and then they were digested by Bsm I and Eco R V to get long probes. We could get MLPA probes by restriction enzyme digestion, and this method could be used to produce a large amount of long MLPA probes.For the probe verification, we cloned 17 positive control vectors, each contained the hybridization DNA sequences of the left and right MLPA probe. After a single-targeted and multi-targeted PCR amplification and ABI3500 capillary electrophoresis analysis, all the short and long MLPA probes were verified to have high sensitivity and targeting specificity. Only 1 ml of M13 phage-derived probes can be used to 100, 000 times of MLPA reactions and the advantage in inexpensiveness is markedly, indicating that the MLPA diagnostic technique for CNS infection pathogen diagnostics could have a huge market potential. In order to get probe diagnostic kits of commercialization in next stage we also did the probe quality analysis. In short, the MLPA kit had high sensitivity, and is fast, easy and inexpensive and had a huge medical diagnostic potential for bacterical CNS infection pathogens.We also designed MLPA probe for detecting the pathogens of encephalitis and meningitis not due to craniotomy. First, in order to ensure every probe has good performance, the reliability and scientific nature of each targeted DNA sequence was verified by relevant research literature. Secondly, we used the Raw probe, MLPA probe Verification and the mfold Web Server such tools to assemble probes. The quality and control parameters reached the standard for the MLPA probe design proposed by MRC-Holland company. As the time problem, I have not enough time during master’s study period to conduct all the probes cloning vecors and related research and development.