Fast Detection Of Mycoplasma Pneumoniae Based On The Tetramolecular G-quadruplex Probe And Graphene Oxide

Objective:Mycoplasma pneumoniae(MP)is one of the important pathogens of community acquired pneumonia,which can induce both the upper and the lower respiratory infections.In recent years,the incidence of MP infection increased significantly.In China,the incidence of MP infection exceeds 15%,and the situation is more serious in some provinces,up to 20%-30%,which has greatly affected people's life quality and health.Although currently there are many diagnostic methods for MP,most of the clinical symptoms caused by MP infection are not typical,and there is a lack of effective early diagnosis methods in clinical practice,Therefore,it is particularly vital to develop an early,rapid and highly specific method for the diagnosis of MP infection to improve the cure rate,reduce the antimicrobial drug abuse,relieve the pain and alleviate the financial burden of patients.This study based on the fluorescence quenching effect of GO and the specific binding of MP DNA and G-tetrad fluorescent probe to achieve the rapid detection of MP DNA.Methods:In this study,a fluorescence probe that can form a G-tetrad was designed.The 5'end of the probe can form a G-tetrad,and the 3'end marks with FAM,and the DNA sequence near the 3'end can specifically bind to the target MP DNA.When there was no target MP DNA,the free single-strand DNA of the G-tetrad was partially adsorbed on GO,and the fluorescence was quenched.When MP DNA was added into the system,the single-stranded part of the G-tetrad and MP DNA were specifically combined to form a double-stranded structure,the G-tetrad was detached from GO and the fluorescence signal was restored.By monitoring the intensity of fluorescence signal,MP was quickly tested.Results:In this study,the fluorescence quenching characteristic of GO was utilized to construct a DNA biosensor detection microplatform through the signal transduction effect of G-tetrad,which was used for the detection of specific conservative DNA sequences of MP.In the absence of the target MP DNA,the fluorescence of the G-tetrad fluorescence probe could be quenched by GO within 2 min,and the quenched efficiency reached over 90%.When the target MP DNA was added into the system,the fluorescence signal of the G-tetrad fluorescence probe was restored,the fluorescence signal recovery value reached the equilibrium state within 10 minutes,and the fluorescence signal recovery rate was as high as 60%of the original fluorescence within one minute.The detection method was successfully used to detect the target MP DNA.The linear range of detection was 5-300 nM,the linear regression equation between fluorescence intensity F and the concentration of the target MP DNA c was F=20.48+0.49 c(R~2=0.998),and the detection line was 3.96 nM,which could realize the detection of the target MP DNA with low concentration.In the specificity experiment,we detected the target MP DNA as well as a single mismatch base,three mismatch bases and random DNA sequence.The changes in fluorescence intensity were 6.06,4.77,3.43 and 1.27 times of the control group,respectively.The fluorescence intensity of the target MP DNA could be clearly distinguished.Conclusions:by optimizing the detection conditions,verifying the feasibility and collecting clinical samples,a simple,rapid,sensitive and highly specific diagnostic method was established,which can realize the early detection of MP and play a positive role in the development of a new diagnostic method for MP.