Construction Of The Method Based On Quantitative Detection Of Sex Chromosome For Non-invasive Screening Of Sex-linked Genetic Diseases

With the development of society and medice, the infectious disease has been effectivelycontrolled. However, hereditary diseases have gradually be the main cause of mortality ofthe infant. Up to date, there have been more than300kinds of sex-linked genetic diseasesbe found, such as hemophilia, color blindness and renal diabetes. Sex-linked geneticdiseases not only affect the patient’s life, but also pose the great burden on the society. Theearly indenficaition of the sex of fetus is able to achieve the purpose of prenatal diagnosisof sex-linked genetic diseases, which undoubtedly has important significance on thereducing the social burden.Currently, prenatal diagnosis has been an issue that must be performed in majorhospitals of china. The diagnostic methods are as follows: chorionic villus sampling,amniocentesis, percutaneous umbilical blood sampling, early amniocentesis, an ultrasonicdiagnosis, fetal biopsy and so on. However, there are many shortcomings, such as easilyleading to miscarriage and infection, low sensitivity and accuracy and so on.In the past few years, the reaseachers have made large number of research on maternalplasma fetal genetic material, and Y chromosome-specific DNA sequences in maternalplasma were confirmed in the fetal maternal plasma for the first time. Currently, cell freefetal DNA have been used for the detection and analysis of fetal RhD blood group,screening for pregnancy-related diseases, chromosomal disease. In recent years, a newtechnology based on the real-time quantitative PCR, called MGB-TaqMan real-time PCRhave been developed, which has a low background fluorescence, higher resolution, strongerhybridization specificity, etc., which is more suitable for the detection of low abundanceDNA, such as cell free fetal DNA.Objectives:Construct a MGB-TaqMan real-time quantitative PCR for the detection and analysis of cffDNA in maternal plasma and for early identification of fetal gender for sex-linkedgenetic diseases.Methods:1. Synthesize X, Y chromosome-specific primers and probes; optimize the PCRreaction conditions, such as pair of primers, probe concentration and Tm for the newlydeveloped method; two cases (one case was male, another female) were used to conformthe optimum reaction conditions of PCR.2. Mixed different proportions of standard genomic DNA from male and female tosimulate the situation of cell free fetal DNA in maternal plasma; evaluated the sensitivity,accuracy and other indicators by the above mixing template.3. Selected50cases outpatient pregnancy specimens with gestational age of16-20weeks randomly and20healthy non-pregnant female specimens to validate and evaluate thenew methods and understand their ability to detect clinical specimens.Results:1. Optimal reaction conditions for real-time quantitative PCR were as follows:0.4M of primer,0.1M of probe, Tm60℃.2. The results of sensitivity showed that the lower limit of the amount of maternalDNA template is50pg. When the amount of template DNA in maternal was between0.5-50ng, abundance of detection for Y chromosome was1%, which indicated that theabundance of fetal DNA was2%; when the amount of template DNA in maternal wasbetween0.05-0.5ng, detecting abundance of Y chromosome was2-4%, which indicatedthat the abundance of fetal DNA was4-8%. When there was no maternal DNA, lower limitof Y chromosome template was0.5pg.3. The results of quantitative detection showed that the concentration of plasma DNAin normal women is up to1.75ng/mL, a minimum of0.50ng/mL, the average value of1.13ng/mL; the concentration of plasma DNA in pregnant women was up to6.50ng/mL, thelowest was0.25ng/mL, an average of3.38ng/mL. The mean concentration of pregnantwomen was2.25ng/mL higher than that of normal women with a statistically significantdifference (P <0.05).4. The results of the detection of49cases fetal sex showed that17cases were maleand32cases were female in the initial experiment, which is not completely consistent withthe sex of fetus after birth. However, after increasing the amount of initial DNA template (> 50pg), the results showed23males and26females. The accuracy was100%.Conclusions:1. The MGB-TaqMan real-time PCR technology for analysis of the fetal DNA inmaternal plasma was successfully constructed. And it could identificate the fetal sex in theearly and provides a non-invasive prenatal screening method for sex-linked geneticdiseases.2. The situation of low abundance of free maternal plasma fetal DNA wassuccessfully simulated by mixing different proportions of standard genomic DNA frommale and female.3. The results of the sensitity analysis showed a high sensitity of the new method. Thelower limit of the amount of maternal DNA template is50pg. When the amount of templateDNA in maternal was between0.5-50ng, abundance of detection for Y chromosome was1%, which indicated that the abundance of fetal DNA was2%. When there was no maternalDNA, lower limit amount of Y chromosome template was0.5pg.4. The MGB-TaqMan real-time PCR technology for analysis of the fetal DNA inmaternal plasma showed that the accuracy was100%. And the plasma DNA levels inpregnant women is significantly higher than normal women.