Detection of analytes and nucleic acids

Abstract

Methods of detecting at least one analyte and at least one nucleic acid in a sample are provided. Reagents for carrying out the methods are also provided.

1 - 32 . (canceled) 
     
     
         33 . A method for detecting and correlating the amount of at least one protein target analyte and at least one target nucleic acid in a cell, comprising:
 a) lysing the cell in a multifunctional lysis buffer to produce a cell lysate, wherein the cell lysate contains at least one protein target analyte and at least one target nucleic acid;   b) incubating the cell lysate with:
 (i) at least one proximity detection probe set, wherein each proximity detection probe set comprises at least two proximity detection probes, and wherein each proximity detection probe comprises at least one oligonucleotide moiety and at least one analyte binding moiety that is capable of binding the at least one protein target analyte, under conditions allowing interaction between the first and second proximity detection probes and the cell lysate; 
   c) detecting and quantitating the protein target analyte by detecting the interaction between the first and the second proximity detection probes using a first real-time quantitative polymerase chain reaction using a first detector probe having a first label to form a first detectable amplicon;   d) detecting and quantitating at least one target nucleic acid by a second real-time quantitative polymerase chain reaction using a second detector probe having a second label to form a second detectable amplicon,   wherein the protein target analyte and the target nucleic acid are both detected and quantitated in the same vessel; and   e) correlating the quantity of the protein target analyte detected with the quantity of the target nucleic acid detected.   
     
     
         34 . The method of  claim 33 , wherein the target nucleic acid is a RNA or a DNA. 
     
     
         35 . The method of  claim 34 , wherein the RNA is a mRNA, a tRNAs, a snRNAs, a rRNAs, a retroviruse, a small non-coding RNA, a microRNAs, a polysomal RNAs, a pre-mRNAs, an intronic RNA, and a viral RNA. 
     
     
         36 . The method of  claim 35 , wherein the target nucleic acid is an RNA, the method further comprising reverse transcribing the RNA prior to PCR amplification. 
     
     
         37 . The method of  claim 34 , wherein the DNA is genomic DNA, plasmid DNA, phage DNA, nucleolar DNA, mitochondrial DNA, chloroplast DNA, cDNA, synthetic DNA, yeast artificial chromosomal DNA (“YAC”), bacterial artificial chromosome DNA (“BAC”), extrachromosomal DNA, and primer extension products. 
     
     
         38 . The method of  claim 33 , further comprising normalizing the quantity of target nucleic acid to a nucleic acid normalizer control and normalizing the quantity of the protein target analyte to a protein analyte normalizer control. 
     
     
         39 . The method of  claim 38 , further comprising using a different label each to detect the nucleic acid normalizer control, the target nucleic acid, the proximity detection probes used to detect the protein analyte normalizer controls, and the proximity detection probes used to detect protein target analyte. 
     
     
         40 . The method of  claim 33 , wherein the step c) comprises multiplex real-time polymerase chain reaction. 
     
     
         41 . The method of  claim 33 , wherein the multifunctional lysis buffer is capable of lysing the cell without substantially degrading the target nucleic acid and while maintaining adequate protein target analyte structure such that one or more proximity detection probes is able to bind the protein target analyte in the lysate if present. 
     
     
         42 . The method of  claim 33 , wherein the multifunctional lysis buffer comprises at least one chemical selected from the group consisting of NDSB-201, LDAO, CHAPS, DEDTAB, Zwittergent 3-10, and CAPSO. 
     
     
         43 . The method of  claim 33 , wherein the cell is from a biological sample selected from a group consisting of prokaryotic cells, eukaryotic cells, tissue samples, viral particles, bacteriophage, infectious particles, pathogens, fungi, food samples, bodily fluids including mucus, blood, plasma, serum, urine, saliva, and semen, water samples, and filtrates from water and air. 
     
     
         44 . The method of  claim 33 , wherein the interaction between the first and the second proximity detection probes is selected from hybridization or ligation of the first and second oligonucleotide moieties of the first and second proximity detection probes. 
     
     
         45 . The method of  44 , wherein the interaction between the first and second proximity detection probes comprises ligation of the first and second oligonucleotide moieties, and wherein the cell lysate is further incubated with:
 ii) at least one splint oligonucleotide; and   iii) at least one ligase; such that at least one ligated proximity detection probe set is formed in the presence of the at least one protein target analyte; and   
       the detection step comprises detecting the at least one ligated proximity detection probe set. 
     
     
         46 . The method of  claim 44 , wherein the interaction between the first and second proximity detection probes comprises hybridization of the first and second oligonucleotide moieties to form a hybridized proximity detection probe set and the detecting step comprises detecting the at least one hybridized proximity detection probe set. 
     
     
         47 . The method of  claim 33 , wherein the detection, quantitation and correlation of the at least one protein target analyte and at least one target nucleic acid analyte is performed in a multiwell plate. 
     
     
         48 . The method of  claim 33 , wherein more than one protein target analytes and more than one target nucleic acids are detected simultaneously in a single detection reaction, the detection reaction comprising using a different label each to identify each proximity detection probe set for each of the protein target analytes and using a different label to identify each of the target nucleic acids. 
     
     
         49 . The method of  claim 48 , wherein five different protein target analytes and five different target nucleic acids are detected in a single detection reaction using five different proximity detection probe sets and ten different labels to separately identify the different real-time quantitative PCR products. 
     
     
         50 . The method of  claim 33 , wherein the detector probes labels are selected from dyes, quenchers, fluorescein dyes, intercalating labels, and combinations thereof.