HPV E6, E7 MRNA Assay and Methods of Use Thereof

Abstract

Provided is an HPV E6, E7 mRNA assay, referenced herein as the “In Cell HPV Assay,” that is capable of sensitive and specific detection of normal cervical cells undergoing malignant transformation as well as abnormal cervical cells with pre-malignant or malignant lesions. The In Cell HPV Assay identifies HPV E6, E7 mRNA via in situ hybridization with oligonucleotides specific for HPV E6, E7 mRNA and quantitates the HPV E6, E7 mRNA via flow cytometry. The In Cell HPV Assay can be carried out in less than three hours directly from liquid-based cervical (“LBC”) cytology specimens. The In Cell HPV Assay provides an efficient and highly sensitive alternative to the Pap smear for determining abnormal cervical cytology.

1 - 55 . (canceled) 
     
     
         56 . A method of detecting a precancerous cell in a cellular sample obtained from a subject, the method comprising:
 fixing and permeabilizing the cells of the cellular sample to produce a prepared cellular sample;   contacting the cells of the prepared cellular sample with an oligonucleotide probe for a nucleic acid analyte expressed by the precancerous cell;   imaging the contacted cells; and   detecting the nucleic acid analyte present in the imaged cells to detect the presence or absence of the precancerous cell in the cellular sample.   
     
     
         57 . The method according to  claim 56 , wherein imaging the cells is performed on a slide. 
     
     
         58 . The method according to  claim 56 , wherein the nucleic acid analyte is an expression product of an oncogene. 
     
     
         59 . The method according to  claim 58 , wherein the oncogene is a viral oncogene. 
     
     
         60 . The method according to  claim 59 , wherein the viral oncogene is an HPV oncogene. 
     
     
         61 . The method according to  claim 60 , wherein the HPV oncogene is HPV E6 or HPV E7. 
     
     
         62 . The method according to  claim 56 , further comprising contacting the cells of the prepared cellular sample with an antibody. 
     
     
         63 . The method according to  claim 56 , further comprising contacting the cells of the prepared cellular sample with a fluorescent nuclear stain. 
     
     
         64 . The method according to  claim 56 , wherein the oligonucleotide probe comprises a detectable label. 
     
     
         65 . The method according to  claim 64 , wherein the detectable label comprises a fluorophore. 
     
     
         66 . The method according to  claim 56 , further comprising signal amplification using an amplifier to detect the oligonucleotide probe. 
     
     
         67 . The method according to  claim 66 , wherein the signal amplification comprises branched DNA (bDNA) signal amplification and the amplifier is a component of the bDNA signal amplification procedure. 
     
     
         68 . The method according to  claim 65 , wherein the signal amplification comprises tyramide signal amplification (TSA) and the amplifier is a component of the TSA procedure. 
     
     
         69 . The method according to  claim 56 , wherein the method further comprises obtaining the cellular sample from the subject. 
     
     
         70 . The method according to  claim 56 , wherein the cellular sample is a liquid cellular sample. 
     
     
         71 . The method according to  claim 70 , wherein the cells of the prepared liquid cellular sample are applied to a slide. 
     
     
         72 . The method according to  claim 56  wherein the cellular sample comprises tissue. 
     
     
         73 . The method according to  claim 72 , wherein the tissue is an epithelium. 
     
     
         74 . The method according to  claim 56 , wherein the fixing and permeabilizing comprises contacting the cells of the cellular sample with a composition comprising a fixation reagent and a permeabilization reagent. 
     
     
         75 . The method according to  claim 56 , wherein the detecting comprises quantifying the nucleic acid analyte present in the imaged cells on a per cell basis and the precancerous cell is detected when the quantified per cell amount of nucleic acid analyte is above a predetermined cut-off point. 
     
     
         76 . The method according to  claim 75 , wherein the predetermined cut-off point is 200 copies of the nucleic acid analyte per cell. 
     
     
         77 . The method according to  claim 56 , wherein the cells contacted with a cocktail of oligonucleotide probes. 
     
     
         78 . The method according to  claim 77 , wherein the cells of the prepared cellular sample are contacted with a cocktail of antibodies. 
     
     
         79 . The method according to  claim 56 , further comprising determining the percentage of cells of the cellular sample expressing the nucleic acid analyte. 
     
     
         80 . The method according to  claim 56 , wherein the imaging comprises microscopic imaging. 
     
     
         81 . The method according to  claim 80 , wherein the microscopic imaging comprises confocal imaging. 
     
     
         82 . The method according to  claim 56 , wherein the detecting is on a per cell basis. 
     
     
         83 . The method according to  claim 56 , wherein the precancerous cell is a precancerous squamous cell.