Establishment And Application Of AZF And Three Methods For Rapid Detection Of Chromosome Number Aberrations

Objective: Chromosome aberration(chromosomal aberration)is divided into number of chromosome aberration and chromosomal structure distortion,which can lead to male infertility and spontaneous abortion and birth defects.Y chromosome microdeletion(Azoospermia factor(AZF)and crohn's syndrome(47,XXY)are common genetic factors causing male infertility.Down syndrome(21-trisomy)and Turner syndrome(45,X)are common causes of spontaneous abortion or birth defects.In the first part,the research status of AZF and chromosome number aberration was systematically and comprehensively introduced,and the traditional diagnosis methods of AZF and chromosome number aberration as well as the new molecular biological diagnosis methods were introduced,thus laying a good theoretical foundation for the rapid detection of chromosome aberration.In the second part,in order to solve the defects of traditional detection technology,a four-fold experimental system was established for the rapid simultaneous detection of two diseases in 40 cases of AZF and 20 cases of 47 and XXY patients collected from the male outpatient department,providing an important tool for the genetic detection of male infertility.In the third part,the High Resolution Melting Analysis(HRM)was used to rapidly diagnose 47,XXY diseases in 78 cord blood samples and 20 dry blood samples during the neonatal period,and its clinical application was evaluated.The fourth part studies and analyzes the application evaluation of HRM technology in the rapid detection of X samples in 3 cases of 21-trisomy and 3 cases of 45 amniotic fluid cells.Methods: In the first part,literature on AZF and chromosome number aberration was searched,queried and sorted from Pubmed database,and the research status and diagnostic methods were summarized.In the second part of the study,a quadruplex system based on qPCR and HRM technology was established and optimized.Rapid simultaneous detection was performed on 40 cases of AZF patients and 20 cases of 47 XXY male infertility patients,and the effectiveness of the method was evaluated by analyzing and diagnosing the sensitivity and specificity.In the third part,HRM technology was used to rapidly diagnose 47,XXY in 78 cases of cord blood samples and 20 cases of dry blood samples in the neonatal period,so as to evaluate the effectiveness of this method.Three methods for DNA extraction of neonatal dried blood samples were compared.In the fourth part,HRM technology was used to rapidly detect X samples from 3 cases of 21-trisomy and 3 cases of 45 amniotic fluid cells to evaluate the effectiveness of the method.Results: Firstly,gel electrophoresis is widely used in traditional AZF detection,but it is inefficient,cumbersome and easy to be contaminated.Karyotype analysis is the gold standarddetection method for chromosome number aberration,but the detection time is long(7-14 days)and requires professional operation,so clinical application is limited.After comparing the advantages and disadvantages of various current diagnostic techniques.Based on qPCR and HRM,a new technique for diagnosing AZF and chromosome number aberration is proposed.Secondly,the detection results of qPCR and HRM system on 40 cases of AZF patients and 20 cases of 47 XXY male infertility patients showed 100% diagnostic sensitivity and specificity.The lower detection limit of the experimental DNA template amount was 2.5ng.Third,HRM technology successfully screened and distinguished normal male and normal female samples from 78 umbilical cord blood samples and 20 cases of dry blood samples.Unfortunately,47,XXY cannot be detected in these samples due to limited sample collection.A comparison of the three methods for DNA extraction of dried blood samples showed no significant difference in the results of DNA extraction using standard commercial kits and boiling methods.Finally,HRM technique was successfully used to detect amniotic fluid in 3 21-trisomic samples,while only 2cases were successfully detected in 3 cases,45 cases and X samples,and 1 case was not successfully differentiated.Conclusion: On the basis of qPCR and HRM technology,this study proposed and established an experimental technology that can rapidly screen AZF and 47,XXY,and preliminarily completed the application evaluation of this technology in three clinical fields,namely male infertility,neonatal abnormality and amniotic fluid cell detection,using HRM.The results show that the technique has the advantages of simple operation,rapid detection,accurate results and high flux.It provides a powerful tool for the rapid diagnosis of AZF and chromosome number aberration.