Improved Loop-Mediated Isothermal Amplification Technique And Its Application In Detection Of Mycoplasma Pneumoniae

Mycoplasma pneumoniae(Mycoplasma pueumoniae Mp)is the primary pathogen of atypical pneumonia in children and adolescents.The common clinical manifestations of respiratory symptoms,such as fever,fever,asthma,may also cause a variety of pulmonary complications,such as encephalitis,skin damage and gastrointestinal involvement and other symptoms,if untreated,severe cases can be life-threatening.Nucleic acid detection is an effective and accurate method for detecting the pathogen of infectious diseases.Commonly used nucleic acid detection methods are based on PCR as a platform for detection.However,PCR-based variable temperature nucleic acid amplification and detection technology cannot be separated from expensive detection instruments in the detection process,which is not conducive to popularization and application in grassroots detection stations and remote areas.The isothermal amplification technology to get rid of dependence on equipment,with good prospects for development in the detection of various pathogens in.The loop-mediated isothermal amplification method is a highly efficient and sensitive nucleic acid amplification method.The method is simple,time-consuming and does not require sophisticated equipment and equipment.It only requires a water bath to complete the reaction.In this study,we used the conserved P1 gene of Mycoplasma pneumoniae as the target and designed corresponding primers to establish a loop-mediated isothermal amplification(LAMP)detection method for Mycoplasma pneumoniae.A hairpin-primer-mediated isothermal amplification(HPIA)technique was established by constructing a novel stem-loop structure primer to achieve rapid and sensitive detection of Mycoplasma pneumoniae.The introduction of accelerated primers on the basis of HPIA accelerated the progress of the HPIA detection technique,and improved the detection of loop-mediated internal primers to achieve quantitative detection of loop-mediated isothermal amplification.The main research contents and results are as follows:1.Detection of Mycoplasma pneumoniae conventional LAMP method.Using M.pneumoniae P1 gene as a target sequence,two internal primers and two external primers were designed to perform an amplification reaction at 65 ? The reaction results after electrophoresis were observed by a gel imaging system.While using the fluorescent dye calcein(Calcein 50 uM,MnCl2 0.5 mM),SYBR Green achieve visual inspection Mycoplasma pneumoniae.2.HPIA detects mycoplasma pneumoniae.A new type of stem-loop structure primer was constructed and a hairpin-primer-mediated isothermal amplification(HPIA)technique was established.Only one pair of primers was needed to achieve rapid and sensitive detection of Mycoplasma pneumoniae.A pair of PCR primers was designed using the P1 gene of M.pneumoniae as a target sequence.A hairpin stem loop was added to its 5' to form a novel stem-loop structure primer,and a hairpin primer-mediated isothermal amplification detection technique was constructed.The minimum detection limit of this method is 100 zmol,which is equivalent to the sensitivity of the traditional LAMP method,and it has good specificity.In addition,we designed a pair of accelerated primers using the P1 gene of M.pneumoniae as the target sequence to accelerate the direction of the extension of the primer and the direction of extension of the stem primer to accelerate the reaction process of the HPIA assay.The results showed that the amplification reaction time for the addition of accelerated primers was significantly shortened and showed strong specificity.3.Improve the traditional loop-mediated internal primers to achieve a preliminary exploration of loop-mediated quantitative detection of isothermal amplification.The loop-mediated amplification was quantitatively detected by introducing a loop to mediate the non-complementary 3 bases of the double dumbbell structure and blocking the self-polymerization reaction of the double dumbbell structure.We designed the experiment by adding bases or introducing mutant bases at the 5' end of the inner primer.The results of electrophoresis experiments showed that the bands had a certain degree of aggregation,but the trapezoidal bands were still evident,indicating that the loop-mediated quantification research needs further study.