Research On Key Technologies Of On-site Diagnosis Of Infectious Diseases And Its Application

In this study, in order to meet the demand for on-site diagnosis of infectiousdiseases, for example on-site nucleic acid extraction, selecting the on-site rapidmolecular diagnostic methods, establishing on-site rapid detection technologyplatforms, We carried out a lot of research and applied the research results in on-sitediagnosis of infectious diseases.Loop-mediated isothermal amplification (LAMP) is a novel in vitro nucleic acidamplification technology established by Notomi et al in2000. The technology use4-6specific sequence primers to identify6-8specific target area. With the help of a DNApolymerase with strand displacement characteristics can quickly, efficiently andspecifically amplify the target sequence at a constant temperature in a short time. Alarge number of target DNA synthesis with by-product production of whitemagnesium pyrophosphate sedimentation. This feature can make the LAMP reactionprocesses been judged negative or positive directly by monitoring turbidity of thereaction. PCR through two primers identified two separate areas of the targetsequence, while the LAMP by identifying the6-8target sequence independent regions.LAMP has a strong specificity compared to PCR, and10-100times can be comparedto sensitivity than conventional PCR, commensurate with the sensitivity offluorescence quantitative PCR. But LAMP amplification process can be carried outunder constant temperature conditions, a stable heat source equipment (such as awater bath or in a metal bath) will be able to meet requirements, testing costs aresignificantly reduced. Since the advent of LAMP technology, it has been widely usedin detection of microorganisms such as bacteria, fungi, viruses, genetic diseasediagnosis, early judging of gender and other fields.Easy and quick LAMP method requires simple and rapid nucleic acid extractionkit to match, then you can give full play to their advantages. Currently on the marketthere are two main types of virus nucleic acid extraction Kit, one is fully automatednucleic acid extraction device, because it is expensive, bulky, requiring professionalmaintenance and mainly used in large hospitals and state laboratories. Another is the common nucleic acid extraction Kit, because of its need of centrifuge, specializedlengthy complicated operation, and mainly used in scientific research units in thecountry. The basic unit is often the starting point for infectious disease outbreaks, ifnucleic acid diagnostic carrying out in situ will play a positive role for prevention andcontrol of infectious diseases. The nucleic acid extraction is the first step inprevention and control. The demand of in situ or basic units in nucleic acid extractionkits are fast, easy to operate, easy to use, non-instrumentation, low professional needs.We have developed nucleic acid extraction kits to meet the demand of in situ or basicunits. This kit is simple to operate, without any equipment, stored at room temperature,can effectively prevent cross-contamination, low degree of specialization needs,without advance with liquid, without pipette, high purity extraction and completedwithin15minutes. Applies to swallow and anal swabs, sputum and other samples ofvirus DNA/RNA extraction.LAMP technology can amplify the target gene for1010times within a short time,which is100-1000times than PCR. Efficient amplification also brings another bigproblem, aerosol pollution, which This is also the LAMP technology promotionencountered the biggest problem. Aerosol pollution caused false positive and falsepositive is very serious. This is also a LAMP researchers encountered every trickyquestion. How to avoid aerosol pollution is also common problems to solve.Laboratory division is widely used as a means, but can not fundamentally solve theproblem. We propose a method to control the amplified product from the source, canbe very good to solve the problem of the formation of aerosols. We invented a sealant,which was added before the experiment as solid. During the reaction, the liquid can bewell prevented formation of aerosols. After completion of the reaction becomes solid,the amplified product is fully enclosed in the tube end. This put an end to theformation of volatile aerosols and the formation of false-positive and can be a goodsolution to false positives caused by amplified products.There are several ways to determine the LAMP reaction results: electrophoresis,SYBR Green Ⅰ staining, precipitation method, turbidimetric method, calceinstaining, hydroxy naphthol blue(HNB) staining and the like. These methods have onething in common: all are through indirect methods to determine LAMP results, theamplified products cannot be directly determined. If they have non-specificamplification, these detection methods are not known. This LAMP Technology suffered another big problem. How to directly detect the amplified product hasbecome a hot research. We get inspiration from the TaqMan probe fluorescencequantitative PCR technique. The TaqMan probe introduced into the LAMP technology,enabling direct detection of the amplified product, it solves the problem ofnon-specific amplification fundamentally. LAMP based on the TaqMan probe methodhas great application value and commercial value.Another big problem faced by the LAMP method is to achieve absolutequantification, some viruses in clinical testing require absolute quantification. LAMPmethod to move towards future clinical applications, it is necessary to overcome thisproblem. LAMP amplification process is complex, amplified products of differentlengths, nor a complete mathematical model to date. There is no mathematicalformula to derive support for LAMP quantitative applications. We found that for somecertain concentration of template, LAMP is very good reproducibility in the test. Forother concentrations of template, its reproducibility becomes irregular. Summary is, atconcentrations above105copies LAMP can achieve absolute quantitative, and R2isabove0.99. When the concentration is105copies or less, you cannot achieve absolutequantification. The reason why there is such a result, we presume that LAMP complexreaction principle-related. The lower concentration of template randomness reactionbecomes large, resulting in low reproducibility of the template concentrationdecreases.After solving the following problems: Sample pretreatment problem, LAMPeasily contaminated problem, non-specific amplification problems, quantitativeproblems, we established a rapid detection platform based on loop-mediatedisothermal amplification technology. Focused on the development of clinical commonpathogenic microorganisms rapid detection kit, and respond to emerging, suddeninfectious diseases. Based on the platform we developed a series of rapid test kit,including diarrhea-associated bacteria detection kit.New, suddenly occurring infectious diseases in recent years gradually increased.In2003, the SARS virus; in2009, the novel influenza H1N1virus; in2010,multi-drug resistant bacteria NDM-1; in2011, the "poison cucumber" caused byEnterohemorrhagic Escherichia coli; in2012, adenovirus type55; in2013, humaninfection H7N9avian influenza virus. Simple, rapid and sensitive detection methodsto control the spread of the epidemic will play a positive role, which allows early detection, early diagnosis and early intervention. We developed the resistant geneNDM-1rapid test kit, Adenovirus type55-14-7rapid detection kit, Kit for rapiddetection of human infection H7N9avian flu virus, with the help of alreadyestablished rapid detection platform based on LAMP technology. Make the materialreserves and technical reserves in response to the outbreak of the epidemic.This research has been devoted to solve the key issues of loop-mediatedisothermal amplification method for the LAMP technology removing obstacles toclinical application, and eventually established rapid detection platform based onloop-mediated isothermal amplification method, which can cope the new, suddeninfectious disease with technical support infectious diseases.