Study On The Key Techniques For Rapid Diagnosis Of Highly Pathogenic Avian Influenza Virus

BackgroundHighly pathogenic avian influenza is a poultry-based infectious disease caused by the type A influenza virus of Orthomyxoviridae.It is characterized as sudden outbreaks,strong infectivity,rapid disease progression,and high mortality rates.Large-scale outbreaks are often accompanied by huge economic losses.Currently,the main method to detect and diagnosis of highly pathogenic avian influenza viruses such as H7N9 and H5N1 is reverse transcription-polymerase chain reaction technology.This technology requires high cost,complicated operation,and specialized persons.The samples need to be collected,concentrated and purified in a preceding procedure,which increases the probability of virus transmission and prolongs the time for obtaining test results.It often takes more than one day to get consequences.The existing detection methods,whether serological or molecular biological detection techniques,responding to the current epidemic situation in which the concentrated outbreak of avian flu alternates with long-term distribution,a large number of recessive infections coexist with a small number of infectious infections,and the common people and the remote rural residents of susceptible populations are somewhat lagging and difficult,which often causes delays in the outbreak and delays in treatment,and even ignites the outbreak.Therefore,it is of great practical significance to develop a method for the detection of highly pathogenic avian influenza viruses that is easy to operate,quick to detect,and relatively inexpensive.ObjectiveOur research group plans to develop a portable diagnostic kit for rapid detection of highly pathogenic avian influenza viruses,involving highly pathogenic avian influenza virus capture,rolling circle amplification,primer generation rolling circle amplification,colorimetric detection and other key technologies.Based on the rolling circle amplification technology,this study explored the rapid amplification method as primer generation rolling circle amplification and colorimetric detect amplification results using metal indicators,laying the technological foundation for rapid detection of highly pathogenic avian influenza viruses.Method?1?Based on isothermal rolling circle amplification method of the padlock probe,the avian influenza virus feature gene sequences were detected.?2?Based on primer generation primer rolling circle amplification method,theavian influenza virus feature gene sequences were detected..?3?DNA amplification is accompanied by the generation of pyrophosphate,and the combination of magnesium and pyrophosphate leads to a decrease in the concentration of magnesium ions,and the metal indicator hydroxy naphthol blue undergoes a color change with the concentration change of magnesium ions,and the reaction result can be directly observed with naked eyes.The colorimetric detection of the amplified product makes it visualized.?4?Quartz crystal microbalance and gel retardation experiments were used to verify the binding of aptamer to H5N1 virus vaccine and recombinant H5N1 HA protein.Results?1?A isothermal rolling circle amplification detection technique based on a padlock probe was successfully established,and optimal loop conditions and optimal amplification conditions were explored.The T4 DNA ligase linkage system was used to determine the sequence concentration of the loops as 250 nM.Amplification was performed using a bst DNA polymerase amplification system and it was determined that 3?L of the template loop was used to amplify for 1 h at 65°C.The entire rolling circle amplification detection based on the padlock probe could be controlled within 3hours.?2?Primer generation rolling circle amplification was designed on the basis of rolling circle amplification.The two sequences designed to generate primers,the primer generation rolling circle amplification results were detected by agarose gel electrophoresis,which showed that the negative and positive controls had significant differences.?3?A colorimetric detection method was successfully established,and the metalindicator hydroxy naphthol blue was used to clearly distinguish whether the system had an amplification reaction,and when there was an amplification reaction,it would show blue color;when there is no amplification reaction,it would show a purple color.The conditions displaying color were optimized,and it was determined that the 8 mM Mg²⁺and 1.6 mM dNTPs were the optimal starting concentrations for hydroxy naphthol blue colorimetric reaction.Under the conditions of pH 9,the presence or absence of amplification reaction was clearly distinguished.?4?Basing on the combination of two ways to capture bird flu virus by magnetic bead-single aptamer and magnetic bead-dual aptamers and two ways to amplication involving rolling circle amplification and primer generation rolling circle amplification,four kinds of programs to detect avian influenza viruses were designed.Then the aptamer and virus binding abilities were preliminary explored and analyzed.However,no suitable aptamers were found at present.ConclusionIn this paper,the isothermal rolling circle amplification was used to detect the feature gene sequences of bird flu virus.Based on the rolling circle amplification,the method of primer generation rolling circle amplification was designed,and then colorimetric detection of amplified products by using metal indicator of hydroxy naphthol blue.And the research has established four methods to detect the bird flu virus,the first program:magnetic beads-single aptamer captures bird flu virus,using rolling circle amplification to signal amplification after releasing the feature gene sequence;the second program:magnetic bead-single aptamer captures avian influenza virus,using primer generation rolling circle amplification to signal amplification after releasing the feature gene sequence;the third program:magnetic bead-dual aptamers capture avian influenza virus,using rolling circle amplification to amplify the second aptamer for signal amplification;the forth program:magnetic bead-dual aptamers capture avian influenza virus and the second aptamer is amplified by using primer generation rolling circle amplification for signal amplification.