Application Of Nucleic Acid Visualization Detection Technology In Bacterial Bloodstream Infection Detection

Bloodstream infection(BSI)refers to the invasion of pathogens such as bacteria,viruses,and fungi into the bloodstream,and rapidly multiply in the blood,causing systemic blood-borne diseases in the body.It is an important cause of death in clinically critically ill patients with high morbidity and mortality.Among them,bacterial bloodstream infection accounts for about 90%of the bloodstream infection pathogens.Blood culture is currently the gold standard for clinical diagnosis of bacterial bloodstream infections and is the most commonly used detection method.However,traditional blood culture takes too long and is susceptible to many factors,and the positive rate is low.The bacterial 16S rRNA is shared by all bacteria and is present in the genome of all bacteria with high conservation and specificity.The detection technology for bacterial 16S rRNA has been widely used,but the products need to be detected by electrophoresis,and the operation is complicated.Real-time fluorescent PCR technology directly monitors the amplified products in real time to avoid the use of electrophoresis,but its cost is high,and it has certain limitations in the application of the primary layer.It is of great significance to develop a rapid,accurate,simple and easy method for the diagnosis of bacterial blood flow infection.In recent years,visual nucleic acid detection technology for bacterial 16S rRNA has developed rapidly.Visual nucleic acid detection technology does not require expensive equipment,easy to operate,and the results are clearly visible,with great advantages.In this study,bacterial PCR universal primers were designed for highly conserved regions in 16S rRNA shared by all bacteria to achieve rapid and simple detection of bacterial bloodstream infection.A biotin-Avidin-System(BAS)specific affinity system was introduced,combined with microsphere enrichment technology,and a rapid and simple mirror based on universal replacement fluorescent primers and microsphere enrichment technology was established by microscopic examination.A new method for detecting bacterial bloodstream infections.A crystal violet-colorless crystal violet lactone(CV-LCV)system was introduced to establish a rapid and simple visual method for detecting bacterial bloodstream infection based on colorless crystal violet lactone.The main research contents and results are as follows:1?Design of universal primers for bacterial PCR.Sequence alignment based on16S rRNA sequences of various bacteria,searching for highly conserved regions of bacteria,designing multiple pairs of bacterial PCR primers for highly conserved regions,and performing Blast specific analysis on the designed primers,which proved that the designed two pairs of primers have High specificity.The electrophoresis results were observed by gel imaging system,and it was proved that the two pairs of primers designed can perform bacterial PCR amplification reaction.The best primers for optimal bacterial PCR were screened to detect bacterial bloodstream infection.2?Microsphere enrichment fluoroscopy was used to detect bacterial bloodstream infection.Universal primers P1 and P2 designed for bacterial 16S rRNA,Probe P1*is the complementary sequence of P1.The quenching group is modified at 5'of the primer P1,the fluorophore is modified at the 3'end of the probe P1*,and the biotin group is modified at the 5'end.Carrying out a PCR amplification reaction,in the absence of a bacterial template,the fluorescent group in P1*and P1 is in contact with the quenching group,and the fluorescence is extinguished.In the presence of a bacterial template,P1 with a quenching group participates in the reaction,releasing a probe P1*with a fluorescent group,and the released fluorescence is captured and enriched by the microbeads,showing strong under the microscope.Fluorescent signal for sample detection,detection limit as low as 10~3cfu/mL.Establish a PCR-microscope to quickly and easily detect microbial detection of bacterial bloodstream infection system to achieve detection of bacterial bloodstream infection.3?Crystal violet visual PCR was used to detect bacterial bloodstream infection.A universal primer was designed for the bacterial 16S rRNA to perform a PCR amplification reaction.The main groove of the dsDNA in the reaction product is combined with the crystal violet present in a small amount in the crystal violet-colorless crystal violet lactone system,so that the equilibrium shifts toward the direction of crystal violet generation,and the system exhibits a distinct blue-violet color,thereby achieving bacteriality,visual detection of bloodstream infections,detection limit as low as 10~3 cfu/mL.The PCR-colorless crystal violet lactone was established to quickly and easily visualize the bacterial bloodstream infection system to detect bacterial bloodstream infection.