Study On The Detection Method Of HER2 Gene Mutations Based On Loop-mediated Isothermal Amplification Technology

The overexpression of HER2 gene is closely related to the occurrence and development of various tumors.In recent years,there have been increasing reports on HER2 mutations and poor prognosis.HER2 S310 F is the most frequent mutation in HER2 gene,and it is located at the key epitope of Pertuzumab binding to the HER2 protein.In addition to promoting the development of cancer,it can also block the binding of therapeutic drugs,resulting in drug resistance.Circulating tumor DNA is a minimally invasive liquid biopsy tool in precision medicine,which in the blood reflected the development of tumors and the changes of genomic in primary and metastatic tumors.Analyzing ct DNA helps monitor drug resistance in patients,avoiding ineffective treatment options and side effects,and saves unnecessary costs for patients.Detection of specific mutations in ct DNA has been written into clinical guidelines for different tumors to predict the efficacy of anti-tumor drugs and concomitant diagnosis.However,due to low concentration,high fragmentation and high mixing with normal DNA fragments,the proportion of ct DNA in patients is quite low and varies greatly.The main challenge to detect tumor-specific mutations in ct DNA is to reach an acceptable level of sensitivity and specificity.At present,FDA has approved concomitant diagnosis for clinical which is mainly based on Next Generation Sequencing(NGS)technology to detect hot spot mutations in ct DNA.However,NGS technology is costly and difficult to afford for many patients.Therefore,it is of great significance to develop a new,sensitive and low-cost detection technology for ct DNA.Loop-mediated isothermal amplification(LAMP)is such a technology.In recent years,a series of LAMP-basedVmethods have been developed,showing great potential in DNA mutation detection,and providing new ideas for disease diagnosis,personalized treatment and precision medicine research.Based on the relatively mature research our research team has conducted on HER2S310 F in the early stage,we carried out a bioinformatics analysis of HER2 mutations and survival,immune infiltration,and gene expression in different tumor patients,and found that HER2 mutations were closely related to the poor prognosis in various tumor patients,among which,patients with HER2 S310 F and patients without HER2 S310 F showed significant differences in overall survival.Based on LAMP,by optimizing the conditions of primer concentration,amplification temperature and the amount of Bst 2.0DNA polymerase,etc,we established a low-cost and high-sensitive LAMP-OSD detection method for HER2 S310 F.At the plasmid level,the detection sensitivity was0.05%.Then,by optimizing the LAMP-OSD method,some bases in the internal primer FIP/BIP were modified on phosphorothioate(PS).After PS modification,the end of double-stranded DNA folded itself to form two stem-loop structures at the extension stage of LAMP amplification,creating additional amplification paths outside the normal amplification path,and improving the amplification efficiency.The established PSLAMP-OSD method was applied to the detection of driver mutation HER2 S310F/Y/A,and the sensitivity was 0.005% at the plasmid level.Compared with LAMP-OSD method,the sensitivity was increased by 10 times,which was more conducive to the detection of low-concentration ct DNA samples.In order to simulate human peripheral blood free DNA,we selected the H460 cell genome as the wild-type template,and the S310F/Y/A mutant plasmid DNA as the mutant template,and randomly interrupted them by ultrasonic fragmentation.By adjusting the duty factor,cycles per burst and treatment time,the length of wild type/mutant DNA fragments was finally consistent with that of ct DNA(160-180 bp).At the DNA fragmentation level(simulated liquid biopsy),the detection standard curve of S310F/Y/A mutation was established,and the detection sensitivity was 0.05%.Then,by designing common primers and three probe sets,optimizing the proportion of probe sets,we realized the detection of different mutations at the same site of HER2 gene using the established detection system.In the LAMP-OSD detection system,different probe ratios showed a good distinction between HER2 S310 F and 310 A,but it was not easy to distinguish HER2 310 Y and wide-type.In contrast,in the PS-LAMP-OSD system,the optimized probe ratio could significantly distinguish the three mutations,and was beneficial for monitoring the drug resistance of tumor patients treated with Pertuzumab.The detection method for driver and drug-resistant mutations HER2 S310F/Y/A using LAMP technology has great potential and is expected to become an economical and effective alternative to the currently used high-cost liquid biopsy technology.In addition,it is of great significance to be used as a companion diagnostic protocol for S310F/Y/A mutation in tumor patients treated with Pertuzumab.Once HER2 S310 F is detected in the patients during treatment,it is necessary to change the treatment plan to avoid missing the treatment opportunity and making illness worse,which helps to achieve maximum clinical benefits.