| Mycoplasma pneumoniae is a common pathogen of respiratory pathogens(RTIS)possessing a strong ability to transmit infection,which can often cause severe upper respiratory pathogen diseases,including community-acquired pneumonia(CAP)occurs in all age groups around the world.In addition,M.pneumoniae can cause damage to the nervous system simultaneously or sequentially,and is associated with a variety of other acute and chronic diseases.Besides,many respiratory viruses,including influenza A virus and influenza B virus,can also cause respiratory infection symptoms such as fever,cough,sore throat and so on,it is difficult to distinguish them in clinic.The accurate diagnosis of pathogens is of great significance to the trea Tment of diseases.The culture method has been the gold standard for the diagnosis of M.pneumoniae,but the test results often take several weeks to get,which is not conducive to the early trea Tment and prevention of diseases.Therefore,other diagnostic methods have been developed,such as the detection of Ig M and/or Ig G by ELISA,the detection of antigen by immunochromatography,and nucleic acid amplification technologies(NAATAs)based on PCR.NAATs based on PCR has gradually become the standard of respiratory tract infection detection due to its high sensitivity,good specificity,and simple operation.For example,during the novel coronavirus(SARS-Co V-2)epidemic period,nucleic acid detection plays a major role in the prevention and control of this epidemic disease.Therefore,it is necessary to develop an efficient and fast detection method.Our group previously invented denaturation bubble-meditated strand exchange amplification(SEA),and we further studied this method based on this,and accelerated the amplification by adding a short high temperature and low temperature process.Speed,because higher temperature is more effective to promote the denaturation of double-stranded DNA(ds DNA)bubbles,so we use a narrow thermal cycle between the denaturation temperature and renaturation temperature to replace the isothermal process,this process is called accelerated chain exchange expansion Increase(ASEA),can complete the detection within 20-30 min.In order to achieve the purpose of simple,rapid and accurate detection of respiratory pathogens,we designed and optimized specific primers for rapid detection of nucleic acid of pathogens,and tested clinical samples to evaluate its practicability.The first part is the design and optimization of ASEA amplification primers.Three different targets were selected in this study.One is M.pneumoniae,whose genetic material is DNA;the other two are influenza A and influenza B viruses,whose genetic material is RNA.The first part is the rapid extraction of nucleic acids from these three different targets.In this study,the magnetic bead method was used to extract the targets.Subsequently,four pairs of specific primers were designed,and then they were evaluated by NUPACK and other methods,and the primers suitable for following study were selected.The results showed that the third pair of the four pairs of primers for M.pneumoniae was the fourth for influenza A virus.The first pair of influenza B virus are the optimal primers.The other nine pairs of primers all have primer dimers or secondary structures,which are not suitable for the following practical applications.Considering that the ASEA detection method has a short high temperature and a short low temperature process,it is necessary to optimize the reaction temperature of the primers.The results show that the optimal temperature for M.pneumoniae is 74?for high temperature and 58?for low temperature.The temperature is74°C in high temperature and 60°C in low temperature.The second part is the rapid detection of nucleic acid of respiratory pathogens.This part studies the feasibility,specificity and sensitivity of M.pneumoniae,influenza A virus and influenza B virus.The results show that ASEA can be used to detect respiratory pathogens,and this method has good specificity.The detection of M.pneumonia can be compared with The other 9 kinds of pathogenic bacteria that also cause similar diseases are distinguished,and 1%of the genomic DNA of M.pneumoniae can be detected from the DNA mixture of multiple pathogens,and the detection limit(LOD)of ASEA is as low as 1.0×10-17M,about120 copy per reaction.The detection of A stream and B stream can also be distinguished from respiratory syncytial virus,adenovirus,etc.,and the detection limit(LOD)of ASEA for RNA can be detected at 1.0×10-15M,about 12000 copy per reaction.Meet the requirements of clinical testing.The third part is the detection of ASEA real samples and its comparison with PCR.The ASEA method was used to detect M.pneumoniae throat swab samples from patients with respiratory pathogens obtained from the Affiliated Medical College of Qingdao University,and the results of this method were compared with those of the hospital.The results showed that the real samples of M.pneumoniae were detected Among the 50 throat swab samples,there were 26 positive samples and 24 negative samples.The test results of this method showed that 25 were positive and 24 were negative,indicated that this method had a false negative result.Further testing of this false-negative result showed that the virus content of this sample was lower than the detection limit of this method,so a false-negative result appeared.The results of this study show that the ASEA method can be used for the detection of respiratory pathogens,with good specificity and sensitivity for DNA and RNA target calibration,and the detection can be completed within 30 minutes;the positive detection rate of real samples of M.pneumoniae is 96.15%,and the negative detection rate is 100%,which can meet the requirements of clinical testing.This method is of great significance for the prevention of influenza and other diseases. |
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