Real-Time PCR-based Newborn Screening Of Severe Combined Immunodeficiency And Congenital Adrenal Hyperplasia

Newborn screening is an important measure for preventive medicine.It can screen child body metabolic changes before clinical symptoms appearance,which could effectively prevent damage to newborn important organs,further protecting newborns normal physical and intelligent development.So,in this study,based on real-time PCR platform,we investigate the gene detection methods of two core panel as Severe combined deficiency(SCID)and Congenital adrenal hyperplasia(CAH),which were published by the American College of Medical Genetics.At the same time,a fast extraction method of blood spot DNA was developed,and the real-time PCR rapid detection method was studied.The first chapter in this study introduced the purpose,significance,process,development status of newborn screening and advantage.Then we explained the concept,principle and application of real-time PCR method.Finally,we revealed our study aim and explained how to apply the real-time PCR method to newborn screening.In chapter two,we confirmed that the chelex 100 method was the simplest,efficient,and less cost way to rapidly extracting blood spots when compared to other three extraction methods.In chapter three,by building a multiplex real-time quantitative PCR system,we quantified TRECs in blood spot samples for SCID screening in newborns.The system consisted of an reference gene RPP30 and a test subject TREC.The relative quantification of TREC using RPP30,and the content of TRECs in 1000,000 peripheral blood mononuclear cells.It can determine the cut-off value of TRECs in normal newborns.If the content of TRECs in the samples test result was lower than the cut-off value,and it was determined as positive samples.We also explored and improved the method for extracting the blood spot DNA.Then this quantitative system was established.In the last chapter,a two rounds of PCR amplification system was described to detect common mutation of CYP21A2 gene and CYP21A2 mutation is the main cause of CAH.Since the CYP21A2 gene is highly homologous to CYP21A1P,it is necessary to achieve specific amplification of the active gene through a preamplification system.The build of preamplification system can amplify the low quality and low concentration blood spot samples with a length of approximately 3.5 kb.The second round of amplification used multicolor melting curve analysis to simultaneously detect 13 common mutation in Chinese.The system detected 524 random blood spot samples,and the genotyping results were consistent with the gold standard Sanger sequencing results in 100%.Detection of 16 clinical samples had an overall accuracy of 100%with the point mutation genotyping when compared with the information provided by the clinical information.The three samples were carrying large fragment deletions that exceeded the scope of the system's detection.Considering its rapidity,ease of use,and accuracy,we concluded that our real-time PCR assay could be detect CYP21A2 mutation in newborn.