METHODS, COMPOSITIONS, AND DEVICES FOR THE RAPID DETERMINATION OF FETAL SEX
The present disclosure provides methods, compositions, and devices for the rapid and direct detection of the sex of a fetus. The disclosure also provides methods, compositions, and devices for detecting fetal nucleic acids in biological samples (e.g., blood, cervical mucus, or urine).
1 . A method for determining the sex of a fetus comprising: a) obtaining a biological sample from a subject who is pregnant or suspected of being pregnant comprising fetal nucleic acids; b) performing isothermal amplification on one or more target fetal nucleic acids in the sample to generate reporter molecules if the target nucleic acids are present in the sample; c) detecting the reporter molecules, wherein detection of the reporter molecules indicates the presence of target fetal nucleic acids in the sample; and d) determining the sex of the fetus based on detecting the presence of the target fetal nucleic acids in the sample.
2 . A method comprising: a) obtaining a biological sample from a subject who is pregnant or suspected of being pregnant comprising fetal nucleic acids; b) performing isothermal amplification on one or more target fetal nucleic acids in the sample to generate reporter molecules if the target nucleic acids are present in the sample; and c) detecting the reporter molecules, wherein detection of the reporter molecules indicates the presence of target fetal nucleic acids in the sample.
3 . A method for determining the sex of a fetus comprising: a) obtaining a biological sample from a subject who is pregnant or suspected of being pregnant comprising fetal nucleic acids; b) contacting the sample with a CRISPR/Cas effector protein, a guide RNA, and a labeled reporter DNA molecule, wherein the labeled reporter DNA molecule is cleaved if one or more target fetal nucleic acids are present in the sample; c) detecting a signal produced by the cleavage of the labeled reporter DNA molecule by the CRISPR/Cas effector protein, wherein detection of the signal indicates the presence of the target fetal nucleic acids in the sample; and d) determining the sex of the fetus based on detecting the presence of the target fetal nucleic acids in the sample.
4 . A method comprising: a) obtaining a biological sample from a subject who is pregnant or suspected of being pregnant comprising fetal nucleic acids; b) contacting the sample with a CRISPR/Cas effector protein, a guide RNA, and a labeled reporter DNA molecule, wherein the labeled reporter DNA molecule is cleaved if one or more target fetal nucleic acids are present in the sample; and c) detecting a signal produced by the cleavage of the labeled reporter DNA molecule by the CRISPR/Cas effector protein, wherein detection of the signal indicates the presence of the target fetal nucleic acids in the sample.
5 . A method for determining the sex of a fetus comprising: a) obtaining a biological sample from a subject who is pregnant or suspected of being pregnant comprising fetal nucleic acids; b) performing isothermal amplification on one or more target fetal nucleic acids in the sample; c) contacting the sample with a CRISPR/Cas effector protein, a guide RNA, and a labeled reporter DNA molecule, wherein the labeled reporter DNA molecule is cleaved if one or more target fetal nucleic acids are present in the sample; d) detecting a signal produced by the cleavage of the labeled reporter DNA molecule by the CRISPR/Cas effector protein, wherein detection of the signal indicates the presence of the target fetal nucleic acids in the sample; and e) determining the sex of the fetus based on detecting the presence of the target fetal nucleic acids in the sample.
6 . A method comprising: a) obtaining a biological sample from a subject who is pregnant or suspected of being pregnant comprising fetal nucleic acids; b) performing isothermal amplification on one or more target fetal nucleic acids in the sample; c) contacting the sample with a CRISPR/Cas effector protein, a guide RNA, and a labeled reporter DNA molecule, wherein the labeled reporter DNA molecule is cleaved if one or more target fetal nucleic acids are present in the sample; and d) detecting a signal produced by the cleavage of the labeled reporter DNA molecule by the CRISPR/Cas effector protein, wherein detection of the signal indicates the presence of the target fetal nucleic acids in the sample.
7 . The method of any one of claims 1 to 6 , further comprising determining the sex of a fetus based on the detection of one or more target fetal nucleic acid sequences on the Y-chromosome.
8 . The method of any one of claims 1 to 7 , wherein the target fetal nucleic acid is cell-free fetal DNA.
9 . The method of any one of claims 1 to 7 , wherein the target nucleic acid is a single copy target sequence or a multicopy target sequence.
10 . The method of claim 9 , wherein the multicopy target sequence is present on the Y-chromosome in more than 20 locations, 30 locations, 40 locations, 50 locations, 100 locations, or 1,000 locations.
11 . The method of any one of claims 1 to 10 , wherein the sample is blood, plasma, serum, saliva, urine, and/or cervical mucus.
12 . The method of any one of claims 1 to 11 , wherein the detection of the reporter molecule is detected in less than 30 minutes.
13 . The method of any one of claims 1 to 11 , wherein the detection of the reporter molecule is detected in less than 60 minutes.
14 . The method of any one of claims 1 to 13 , wherein the detection is carried out with an Electrochemical chip, a Graphene, field-effect transistor, a nanopore sense, an SMR sensor, a nanoelectrokinetic chip, or microarray.
15 . The method of any one of claims 1 to 14 , further comprising amplifying the target DNA in the sample by loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), and/or isothermal multiple displacement amplification (IMDA).
16 . The method of any one of claims 1 to 15 , further comprising contacting the biological sample with a preservative composition.
17 . The method of any one of claims 1 to 16 , further comprising contacting the biological sample with a protectant composition.
18 . The method of claim 17 , wherein the protectant composition comprises dextran, trehalose, and/or pullulan.
19 . The method of any one of claims 3 to 18 , wherein the guide RNA comprises more than one crRNA.
20 . The method of any one of claims 3 to 19 , wherein the CRISPR/Cas effector protein is Cas9, Cas12a, Cas 14a/b, and/or Cas 13a/b.
21 . The method of claim any one of claims 16 to 20 , wherein the preservative composition comprises an anti-coagulant (e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), heparin), an antimicrobial (e.g., imidazolidinyl urea), a sugar, and/or an amino acid.
22 . The method of any one of claims 1 to 21 , further comprising enriching the sample for fetal nucleic acids.
23 . The method of claim 22 , 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.
24 . The method of any one of claims 1 to 23 , wherein the fetal nucleic acids are cell-free fetal nucleic acids or genomic fetal nucleic acids from a fetal cell.
25 . The method of any one of claims 1 to 24 , further comprising isolating, enriching, and/or concentrating the fetal nucleic acids.
26 . The method of any one of claims 1 to 25 , wherein the sex of the fetus is determined with at least 90% accuracy.
27 . The method of any one of claims 1 to 26 , wherein the gestational age of the fetus is between 4 weeks and 20 weeks.
28 . The method of any one of claims 1 to 27 , wherein the sample volume is less than 1 ml.
29 . The method of any one of claims 14 to 28 , wherein the preservative is an anti-coagulant (e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(O-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), heparin), an antimicrobial (e.g., imidazolidinyl urea), a sugar, and/or an amino acid.
30 . A device for detecting the presence of one or more target fetal nucleic acids in a biological sample, the device comprising: a lateral flow strip; a detection region on said lateral flow strip comprising a detectable particle or label: a fluid sample comprising a maternal biological sample comprising fetal nucleic acids; wherein said detection region provides a visual colorimetric signal indicating the presence of the target fetal nucleic acid in the fluid sample in less than two hours by capillary flow.
31 . The device of claim 30 , further comprising a CRISPR composition, a preservative composition, an amplification composition, and/or a protectant composition.