Pcr validation tubes

Abstract

This invention relates to a PCR fluorescence reference standard and to a method for manufacturing a PCR fluorescence reference standard. The PCR fluorescence reference standard comprises a fluorophore suspended in a thermoplastic polymer matrix. The PCR fluorescence reference standard of the invention has a greater shelf life than fluorophores dissolved in a solution and can advantageously be used to validate a fluorescence signal obtained in a thermal cycler.

1 . A PCR fluorescence reference standard, comprising a fluorophore suspended in a thermoplastic polymer matrix selected from the group consisting of poly methyl methacrylate (PMMA); polycarbonate (PC); poly oxymethylene (POM); chlorinated polyvinyl chloride (CPVC); and PVC/Acrylic copolymer, 
     
     
         2 . The PCR fluorescence reference standard as recited in  claim 1 , wherein the polymer is PMMA. 
     
     
         3 . The PCR fluorescence reference standard as recited in  claim 1 , wherein the fluorophore is chosen from the group consisting of: aromatic hydrocarbons and derivatives thereof; bis-benzimides and derivatives thereof; coumarin and derivatives thereof; cyanine and derivatives thereof; fluorescent drugs and derivatives thereof; fluorescent proteins and derivatives thereof; naphtalimide dyes; perylene, xanthene and derivatives thereof; 4,6-diamidino-2-phenylindole; oxazole yellow, derivatives thereof and homodimers thereof; thiazole orange, derivatives and homodimers thereof; pyrenyloxytrisulfonic acid; propidium iodide: ethidium bromide: acridine orange; nitrobenzo-2-oxa-1,3-diazole, tetraphenylbutadiene; oxonol fluorescent dyes; 7-Aminoactinomycin D; aminonaphthalimide dyes; and quinolinium 6-(dimethylamino)-2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-1-ethyl perchlorate. 
     
     
         4 . The PCR fluorescence reference standard as recited in  claim 3 , wherein the fluorophore is a conjugate or combination of two or more certified reference materials. 
     
     
         5 . The PCR fluorescence reference standard as recited in  claim 1 , wherein the fluorophore concentration is below 10 −2  mol/L. 
     
     
         6 . A method of validating a thermal cycler, comprising the step of suspending a fluorophore in a thermoplastic polymer matrix selected from the group consisting of: poly methyl methacrylate (PMMA): polycarbonate (PC); poly oxymethylene (POM); chlorinated polyvinyl chloride (CPVC); and PVC/Acrylic copolymer. 
     
     
         7 . The method of validating a thermal cycler as recited in  claim 6 , wherein the thermoplastic polymer matrix is a PMMA matrix. 
     
     
         8 . The method of validating a thermal cyder as recited in  claim 6 , wherein the fluorophore is a certified reference material chosen from the group consisting of: coumarin and derivatives thereof; xanthene and derivatives thereof; cyanine and derivatives thereof; aromatic hydrocarbons and derivatives thereof; fluorescent proteins and derivatives thereof; bis-benzimides and derivatives thereof; fluorescent drugs and derivatives thereof; 4′,6-diamidino-2-phenylindole; oxazole yellow, derivatives thereof and homodimers thereof; thiazole orange, derivatives and homodimers thereof; pyrenyloxytrisulfonic acid; propidium iodide; ethidium bromide; acridine orange; nitrobenzo-2-oxa-1,3-diazole; tetraphenylbutadiene; oxonol fluorescent dyes; 7-Aminoactinomycin D; aminonaphthalimide dyes; and quinolinium 6-(dimethylamino)-2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-1-ethyl perchlorate. 
     
     
         9 . The method of validating a thermal cyder as recited in  claim 7 , wherein the fluorophore is a conjugate or combination of two or more certified reference materials. 
     
     
         10 . The method of validating a thermal cycler as recited in  claim 6 , wherein the fluorophore concentration is below 10 −2  mol/L. 
     
     
         11 . A method for manufacturing a PCR fluorescence reference standard, comprising the steps of:
 (a) providing thermoplastic monomers for making a thermoplastic polymer matrix selected from the group consisting of: poly methyl methacrylate (PMMA), polycarbonate (PC), poly oxymethylene (POM), chlorinated polyvinyl chloride (CPVC), and PVC/Acrylic copolymer;   (b) mixing the thermoplastics monomers with a fluorophore;   (c) carrying out a bulk polymerisation reaction to obtain a thermoplastic polymer matrix comprising a fluorophore suspended substantially uniformly therein;   (d) drying the polymer comprising the suspended fluorophore;   (e) machining the dried polymer comprising the suspended fluorophore; and   (f) polishing the dried polymer comprising the suspended fluorophore.