| Infectious diseases are a class of diseases that keep the international health organization andthe major hospitals and centers of disease control on high alert,usually caused by a specific pathogen.They can spread between people,animals and animals,people and animals.The outbreak and epidemic of infectious diseases constitute a great threat to human life and property.The outbreak of several major infectious diseases in history can be said to be shocking.In a very short period of time,they can be popular in cities around the world,and some serious ones can even paralyze a city,especially diseases caused by respiratory virus.Early prevention and rapid and accurate diagnosisare crucial to the prevention and treatment of infectious diseases.There are many kinds of pathogenic microorganisms that can cause infectious diseases,among them,the most frequent,the most serious harm is the virus.In particular,the emergence and outbreak of SARS,H1N1,H5N1,H7N9,EBOLA and MERS in recent years,let us recognize that there is a long way to go to fight the virus.In this study,the rapid extraction method of nasopharyngeal swab virus nucleic acid was first established.Then the feasibility of polymerase helical reaction amplification RNA was discussed.Then for six respiratory viruses(Influenza a(H1N1),influenza a(H5N1)virus,influenza a(H7N9)virus,influenza a(H3N2),adenovirus,human pneumoconiosis),we established a fast detection method.Finally,kits that can quickly detect the six viruses in one hour were assembled.Nucleic acid extraction has become the most important and basic operation in molecular biology experiment technology,but at present,there are a lot of virus nucleic acid extraction programs,and some are complicated,some are simple.Grass-roots units are often the starting point for outbreaks of infectious diseases.Rapid nucleic acid diagnosis can play a positive role in the prevention and control of infectious diseases,and nucleic acid extraction is the first step in prevention and control.According to the medical conditions at the site or at the base level,the demand for nucleic acid extraction kit is fast,easy to operate,easy to use,no instrumentation,and low professional demand.We developed a rapid extraction solution of nasopharyngeal swab virus nucleic acid from the demand of site nucleic acid extraction.The extraction procedure is simple to operate,not to need any instrument,kept in normal temperature,which can effectively prevent cross contamination and the degree of specialization is low.We don't need fluid preparation in advance.and the fluid can be extracted within 5 minutes.And it can be directly used for subsequent nucleic acid amplification reactions,including conventional PCR,fluorescence quantitative PCR,PSR,LAMP isothermal amplification,etc.The polymerase helical reaction has been successfully used to detect a variety of bacteria and DNA viruses,such as klebsiella pneumoniae,pseudomonas aeruginosa and enterovirus 71,which can all be detected corresponding pathogenic microorganisms which has high specificity.It provides a new convenient and reliable detection method and diagnostic basis for such diseases.In this study,the theoretical feasibility of polymerase helical reaction amplification RNA was discussed first.Because the PCR method requires a thermal cycle,in one of which the temperature is 90 ? to 95 ?,and the reverse transcriptase is inactive at this temperature,and this also determines that the fluorescence quantitative PCR method needs to be divided into two steps to detect RNA viruses.First,reverse transcriptional RNA at lower temperatures,and then the heat cycle amplifies the c DNA,and this will inevitably lead to longer detection time.We were studying reverse transcriptase,we found that in 30 ? to 68 ? of cases,it is still active.This temperature range contains the temperature of the polymerase spiral amplification.In this case,we can do both at the same time,avoiding the trouble of two reactions,therefor save the time.This inference is also confirmed in this study.In this method,the polymerase helical reaction is simpler and more convenient,meanwhile,the application range of polymerase helical reaction is extended.Influenza is the most common respiratory disease which is caused by influenza virus,which has characteristics like rapid propagation and easy outbreak.Seasonal epidemics and regional outbreaks occur in countries all over the world each year.According to the the statistics from world health organization,between 3 and 5 million of the annual infections develop into severe infections,and about 30 to 500,000 people died.It is one of the more common and difficult to control infectious diseases.The biggest problem in treating and preventing influenza is that Influenza viruses are susceptible to antigenic variation,and this allows the virus to escape the host's immune recognition,having initiated outbreaks and epidemics of new types of influenza.Aiming at specific genes for influenza a(H1N1),influenza a(H5N1),influenza a(H7N9),influenza a(H3N2),adenovirus,and human pneumoconiosis,PSR primers were designed.The results were determined by real-time turbidity method and explicit color method.Determining the optimum temperature separately,further experiments showed that the primers had good specificity and no cross reactions with other respiratory pathogens,sensitivity up to 10-100 copies,and it is 10 to 100 times more sensitive than normal PCR.The method is simple,fast,specific and sensitive.It is especially suitable for the application and promotion of site and grass-roots units.In this study,6 kinds of rapid detection kits of respiratory virus were established,which were "storage and transportation at room temperature,open seal,convenient operation and intuitive results".The kit consists of four parts: sampling cotton swab,sample processing tube,test tube and thermostat.After five simple steps of "cotton swab sampling,oscillation cracking,drip sample,constant temperature amplification,observation result".all the steps can be completed within 1 hour.No other equipment required,no fluid prepared,nucleic acid detection can be carried out even in extreme environments where water and electricity are cut off.It is especially suitable for the popularization and application of primary health units. |
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