The Establishment Of A Detection Method For Spinal Muscular Atrophy

Spinal muscular atrophy(SMA)is a complex recessive inherited disease of motor neuron degeneration.It ranks first in childhood fatal genetic diseases,and the surviving patients have serious disease phenotypes,so how to accurately and effectively for the newborns disease screening and forecasts for the severity of treatment plays an important role in the late.At present,SMA detection methods are mostly sequencing,MLPA and dd PCR.The existing detection technologies have their own advantages,but the operation is relatively complicated,the cost of equipment is high,and it is difficult to achieve popularization as a routine screening.Therefore,establishing a kind of quick and easy low-cost detection method for different levels of medical institutions for SMA disease screening is of great significance.In this study,the chromatographic technology with Gold Magnetic Nanoparticles as the core carrier is combined with ARMS-PCR,and the labeled PCR product interacts with the surface antibodies of the Gold Magnetic Nanoparticles in the constructed detection system.It is fixed at the detection line to realize the detection of gene deletion polymorphism and the magnetic detection of gene copy number.In this paper,a homozygous deletion polymorphism detection method of the exon of the pathogenic gene SMN1 was firstly established for screening SMA diseases.At the same time,the copy number quantitative detection method of functional complementary gene SMN2 was established for the evaluation of disease severity.Based on this,the related plasmid DNA of SMN1 and SMN2 genes was established as the positive quality control substance in this experiment,followed by primer design,system establishment and chromatographic system optimization.In this study,a homozygous deletion polymorphism detection technique for SMN1 gene exon was established and its performance was evaluated.The sensitivity of two polymorphism detection sites was 5ng/μL for exon7 and 2.5ng/μL for exon8.In addition,a quantitative magnetic detection method for copy number of SMN2 gene was established and its performance was evaluated,and the standard for quantitative detection of copy number of SMN2 gene was established,the R2 value of the standard curve obtained from the signal value and the copy number is 0.9966.Finally,the constructed SMN1 and SMN2 detection methods were verified by using positive quality control substance,negative control,homozygous deletion plasmid and 50 healthy people,and the accuracy and effectiveness of the polymorphism detection method and quantitative detection method were analyzed by sequencing comparison,the consistency of the two detection methods is 100%.This study is based on the established polymorphism detection method and gene copy number magnetic quantitative detection method of SMA disease,which is simple and convenient to operate,and has strong universality.It provides a new technical idea for the rapid screening of disease and the prediction of disease severity.