Materials and methods for multiplex detection of body fluids

Abstract

The invention pertains to analyzing the levels of DNA methylation at specific genetic loci to detect specific body fluids, for example, vaginal secretions or vaginal epithelial cells, semen or sperms, saliva or buccal epithelial cells, or blood or blood cells. Particularly, the levels of methylation of DNA at the genetic loci corresponding to SEQ ID NOs: 1, 6, 11, and 16, more particularly, SEQ ID NOs: 25, 26, 27, and 28, are used to detect vaginal secretions or vaginal epithelial cells, semen or sperms, saliva or buccal epithelial cells, and blood or blood cells, respectively. The level of methylation at the specific loci can be determined by sequencing of the amplicons produced using specific primers designed to amplify the specific loci. Kits containing the primers and reagents for carrying out the methods disclosed herein are also provided.

1 . A method for identifying a sample as containing or not containing one or more human cells selected from a vaginal epithelial cell, a sperm, a blood cell, or a buccal epithelial cell, the method comprising the steps of:
 a) determining a level of methylation at the genetic loci corresponding to one or more of SEQ ID NOs: 1, 6, 11, and 16, in:
 i) a genomic DNA from the sample, and 
 ii) optionally, a control genomic DNA; 
   b) optionally, obtaining one or more reference values corresponding to the levels of methylation at the genetic loci corresponding to one or more of SEQ ID NOs: 1, 6, 11, and 16; and   c) identifying the sample as:
 i) containing, or not containing, the vaginal epithelial cell based on the level of methylation at the genetic locus corresponding to SEQ ID NO: 1 in the genomic DNA isolated from the sample, 
 ii) containing, or not containing, the sperm based on the level of methylation at the genetic locus corresponding to SEQ ID NO: 6 in the genomic DNA isolated from the sample, 
 iii) containing, or not containing, the blood cell based on the level of methylation at the genetic locus corresponding to SEQ ID NO: 11 in the genomic DNA isolated from the sample, and/or 
 iv) containing, or not containing, the buccal epithelial cell based on the level of methylation at the genetic locus corresponding to SEQ ID NO: 16 in the genomic DNA isolated from the sample. 
   
     
     
         2 . The method of  claim 1 , wherein the sample is processed to separate a cell suspected to be a vaginal epithelial cell, sperm, blood cell, or buccal epithelial cell before the step of isolating the genomic DNA. 
     
     
         3 . The method of  claim 1 , wherein the sample consists of vaginal epithelial cells, sperms, blood cells, buccal epithelial cells, or a combination thereof. 
     
     
         4 . The method of  claim 1 , wherein the levels of methylation at the specific loci in the genomic DNA obtained from the sample is determined by a sequencing analysis, wherein the sequencing analysis comprises the steps of:
 a) isolating the genomic DNA from the sample and optionally, the control sample;   b) treating the isolated genomic DNA with bisulfite;   c) PCR amplifying the genetic loci corresponding to one or more of SEQ ID NOs: 1, 6, 11, and 16 to produce the corresponding one or more amplicons, wherein the PCR amplifying is performed using one or more primer pairs selected from:
 i) SEQ ID NOs: 2 and 3, 
 ii) SEQ ID NOs: 7 and 8, 
 iii) SEQ ID NOs: 12 and 13, and 
 iv) SEQ ID NOs: 17 and 18; and 
   d) determining the sequences of the one or more amplicons; and   e) identifying the sample as:
 i) containing, or not containing, the vaginal epithelial cell based on the sequence of the amplicon corresponding to the genetic locus corresponding to SEQ ID NO: 1 in the genomic DNA isolated from the sample, 
 ii) containing, or not containing, the sperm based on the sequence of the amplicon corresponding to the genetic locus corresponding to SEQ ID NO: 6 in the genomic DNA isolated from the sample, 
 iii) containing, or not containing, the blood cell based on the sequence of the amplicon corresponding to the genetic locus corresponding to SEQ ID NO: 11 in the genomic DNA isolated from the sample, and/or 
 iv) containing, or not containing, the buccal epithelial cell based on the sequence of the amplicon corresponding to the genetic locus corresponding to SEQ ID NO: 16 in the genomic DNA isolated from the sample. 
   
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 4 , wherein the sequencing analysis of the one or more amplicons is performed by a sequencing primer, wherein the sequencing primer is designed based on the sequences of SEQ ID NOs: 1, 6, 11, or 16 and/or the sequences of the regions flanking the genetic loci corresponding to the sequences of SEQ ID NOs: 1, 6, 11, or 16. 
     
     
         7 . The method of  claim 4 , wherein PCR amplification with the two or more primer pairs is performed in one reaction. 
     
     
         8 . The method of  claim 4 , wherein the sequencing primer for sequencing the amplicon corresponding to the sequence of SEQ ID NO: 1, 6, 11, or 16 is SEQ ID NO: 21, 22, 23, or 24, respectively. 
     
     
         9 . The method of  claim 4 , wherein the step c) comprises PCR amplifying the genetic loci corresponding to SEQ ID NOs: 1, 6, 11, and 16 to produce the corresponding amplicons, wherein the PCR amplifying is performed using the primer pairs of
 i) SEQ ID NOs: 2 and 3,   ii) SEQ ID NOs: 7 and 8,   iii) SEQ ID NOs: 12 and 13, and   iv) SEQ ID NOs: 17 and 18; and   
       step d) of determining the sequence of the amplicons corresponding to SEQ ID NOs: 1, 6, 11, and 16 is performed using sequencing primers comprising SEQ ID NOs: 21, 22, 23, and 24, respectively. 
     
     
         10 . A method for determining the level of methylation at genetic loci corresponding to one or more of SEQ ID NOs: 1, 6, 11, and 16, in a genomic DNA isolated from a human cell. 
     
     
         11 . The method of  claim 10 , wherein the cell is suspected to be a vaginal epithelial cell, sperm, blood cell, or buccal epithelial cell. 
     
     
         12 . The method of  claim 10 , wherein the levels of methylation at the specific loci in the genomic DNA obtained from the sample is determined by a sequencing analysis, wherein the sequencing analysis comprises the steps of:
 a) isolating the genomic DNA from the cell;   b) treating the isolated genomic DNA with bisulfite;   c) PCR amplifying the genetic loci corresponding to one or more of SEQ ID NOs: 1, 6, 11, and 16 to produce the corresponding one or more amplicons, wherein the PCR amplifying is performed using one or more primer pairs selected from
 i) SEQ ID NOs: 2 and 3, 
 ii) SEQ ID NOs: 7 and 8, 
 iii) SEQ ID NOs: 12 and 13, and 
 iv) SEQ ID NOs: 17 and 18; and 
   d) determining the sequences of the one or more amplicons.   
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 12 , wherein the sequencing analysis of the one or more amplicons is performed by one or more sequencing primers, wherein the sequencing primers are designed based on the sequences of SEQ ID NOs: 1, 6, 11, or 16 and/or the sequences of the regions flanking the genetic loci corresponding to the sequences of SEQ ID NOs: 1, 6, 11, or 16. 
     
     
         15 . The method of  claim 12 , wherein PCR amplifying with the two or more primer pairs is performed in one reaction. 
     
     
         16 . The method of  claim 12 , wherein the sequencing primer for sequencing the amplicon corresponding to the sequence of SEQ ID NO: 1, 6, 11, or 16 is SEQ ID NO: 21, 22, 23, or 24, respectively. 
     
     
         17 . The method of  claim 12 , wherein the step c) comprises PCR amplifying the genetic loci corresponding to SEQ ID NOs: 1, 6, 11, and 16 to produce the corresponding amplicons, wherein the PCR amplifying is performed using the primer pairs of
 i) SEQ ID NOs: 2 and 3,   ii) SEQ ID NOs: 7 and 8,   iii) SEQ ID NOs: 12 and 13, and   iv) SEQ ID NOs: 17 and 18; and   
       step d) of determining the sequence of the amplicons corresponding to SEQ ID NOs: 1, 6, 11, and 16 is performed using sequencing primers comprising SEQ ID NOs: 21, 22, 23, and 24, respectively. 
     
     
         18 . A kit comprising a primer pair having the sequences of SEQ ID NOs: 12 and 13. 
     
     
         21 . The kit of  claim 18 , further comprising a sequencing primer comprising the sequence of SEQ ID NO: 23. 
     
     
         22 . The kit of  claim 18 , further comprising one or more primer pairs, and optionally, one or more sequencing primers, selected from:
 i) a primer pair having the sequences of SEQ ID NOs: 2 and 3 and optionally, a sequencing primer comprising SEQ ID NO: 21,   ii) a primer pair having the sequences of SEQ ID NOs: 7 and 8 and optionally, a sequencing primer comprising SEQ ID NO: 22, and   iii) a primer pair having the sequences of SEQ ID NOs: 17 and 18 and optionally, a sequencing primer comprising SEQ ID NO: 24.   
     
     
         23 . The kit of  claim 22 , the kit comprising:
 i) the primer pair having the sequences of SEQ ID NOs: 2 and 3,   ii) the primer pair having the sequences of SEQ ID NOs: 7 and 8,   iii) the primer pair having the sequences of SEQ ID NOs: 12 and 13, and   iv) the primer pair having the sequences of SEQ ID NOs: 17 and 18.   
     
     
         24 . The kit of  claim 23 , further comprising the sequencing primers comprising the sequences of SEQ ID NOs: 21, 22, 23, and 24.