Methods, compositions, and kits for determining the sex of a fetus

Abstract

The disclosure relates to methods, compositions, and kits for the early determination of the sex of a fetus. The disclosure also provides methods, compositions, and kits for detecting fetal nucleic acids in biological samples (e.g., cell-free fetal DNA).

What is claimed is: 
     
         1 . A method of determining the sex of a fetus in a pregnant subject, comprising: obtaining a biological sample from the subject; and detecting fetal Y-chromosome nucleic acids in the sample, thereby determining the sex of the fetus. 
     
     
         2 . The method of  claim 1 , further comprising enriching the sample for fetal nucleic acids. 
     
     
         3 . The method of  claim 2 , wherein the enrichment is achieved by separating plasma from whole blood, by selectively capturing fetal nucleic acids from the biological sample, and/or by selectively degrading maternal nucleic acids in the biological sample. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the Y-chromosome nucleic acids are cell-free fetal nucleic acids or genomic fetal nucleic acids from a fetal cell. 
     
     
         5 . The method of any one of  claims 1 - 4 , further comprising isolating and concentrating the fetal nucleic acids. 
     
     
         6 . The method of  claim 5 , wherein the isolating is achieved with a silica column or magnetic beads. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein the sex of the fetus is determined with at least 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% accuracy. 
     
     
         8 . The method of  claim 7 , wherein the performance of the method has been determined in a population of at least 50 or more pregnant subjects. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the gestational age of the fetus is selected from the group consisting of between 4 weeks and 8 weeks, not more than 56, 53, 49, 46, 42, 39, 35, 32, or 28 days or a range defined by any two of the preceding values, 35-53 days, 42-49 days and 42-53 days. 
     
     
         10 . The method of any one of  claims 1 - 9 , wherein the detecting comprises performing microarray analysis, polymerase chain reaction (PCR), or sequencing. 
     
     
         11 . The method of  claim 10 , wherein the PCR is a real-time quantitative PCR. 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein the sample is blood, plasma, serum, saliva, urine, and/or cervical mucus. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the sample volume is selected from the volume of less than 1 ml, 750 μl, 500 μl, 250 μl, 200 μl, 175 μl, 150 μl, 125 μl, 100 μl, 75 μl, 50 μl, and 25 μl, and a range of 25-500 μl, 25-250 μl, 50-125 μl, and 100-250 μl. 
     
     
         14 . The method of any one of  claims 1 - 13 , wherein the biological sample is processed within 1 hour, within 24 hours, or within 48 hours. 
     
     
         15 . The method of any one of  claims 1 - 14 , wherein the biological sample is incubated or mixed with a preservative. 
     
     
         16 . The method of any one of the preceding claims, wherein the biological sample is stored at ambient temperature for at least 12, 18, 24, 36 or 48 hours, 12-48 hours, 18-48 hours, 18-36 hours, or 18-24 hours, before processing, and wherein the biological sample is whole blood. 
     
     
         17 . The method of  claim 16 , wherein the whole blood is capillary blood. 
     
     
         18 . The method of claim any of the preceding claims, wherein the method comprises:
 a) obtaining or having obtained a blood sample from a pregnant subject, wherein the volume of blood is 80-10,000 μl, 80-5,000 μl, or 80-1,000 μl of venous blood, or 80-500 μl, or 80-250 μl of capillary blood; b) mixing the blood sample with a cell-free nucleic acid preservative;   c) storing the blood sample for at least 12, 18, 24, 36 or 48 hours, 18-48 hours, 18-36 hours, or 18-24 hours, at ambient temperature;   d) collecting 40-5,000 μl, 40-2,500 μl, or 40-500 μl of plasma from the venous blood sample, or 40-250 μl or 40-125 μl of plasma from the capillary blood sample;   e) isolating cell-free nucleic acids from the plasma;   f) detecting Y-chromosome DNA in the cell-free nucleic acids by performing quantitative PCR with all or a portion of the isolated cell-free nucleic acids, wherein the gestational age of the fetus is not more than 56, 53, 49, 46, 42, 39, 35, 32, or 28 days, or 35-53 days, 42-49 days or 42-53 days, and wherein the accuracy of determining fetal sex is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% determined in a population of at least 50 or more pregnant subjects.   
     
     
         19 . The method of  claim 18 , further comprising g) determining the sex of a fetus as male based on the detection of Y-chromosome DNA in the cell-free nucleic acids, or female based on the absence of detection of Y-chromosome DNA in the cell-free nucleic acids. 
     
     
         20 . The method of  claim 18  or  19 , wherein the wherein the volume of blood is 80-500 μl of capillary blood, wherein 40-250 μl of plasma is collected, wherein the gestational age of the fetus is 42-53 days and the accuracy of the test is at least 99%. 
     
     
         21 . The method of any one of the preceding claims, wherein the sample is incubated or mixed with a cell-free nucleic acid preservative, wherein the preservative is an anti-coagulant (e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), heparin), an antimicrobial, a sugar, and/or an amino acid. 
     
     
         22 . The method of any one of the preceding claims wherein the sample is a capillary blood sample collected from the upper arm of the subject. 
     
     
         23 . The method of any one of the preceding claims wherein the method comprises decontaminating the sample collection site. 
     
     
         24 . The method of any one of the preceding claims wherein the sample collection site is not a hand or finger. 
     
     
         25 . The method of any one of the preceding claims wherein the method does not comprises decontaminating the sample collection site. 
     
     
         26 . A kit for collecting a biological sample from a pregnant subject for determining fetal sex, the kit comprising a blood collection tube, a lancet or a device for obtaining venous or capillary blood from the subject, a tourniquet, a bandage, an alcohol swab, optionally a nail or skin brush, and instructions. 
     
     
         27 . The kit of  claim 26 , further comprising a decontaminating agent. 
     
     
         28 . The kit of  claim 27 , wherein the decontaminating agent is bleach, an alcohol wipe, chlorhexidine gluconate, hydrogen peroxide, and/or iodine. 
     
     
         29 . The kit of any one of the preceding claims, wherein the instructions provide for sample collection at gestational age of 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. 
     
     
         30 . The method or kit of any one of the preceding claims wherein the subject is a human.