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.

Description

FIELD OF THE INVENTION[0001]The present invention relates to means for validating a polymerase chain reaction (PCR) apparatus, also known as thermal cycler. In addition, the present invention relates to a method for validating thermal cyclers.BACKGROUND OF THE INVENTION[0002]Polymerase chain reaction, hereinafter PCR, is a method of amplifying a DNA target sequence, i.e. a method of producing a large number of copies of a given sequence of DNA, in a relatively short period of time. A PCR reaction solution therefore includes a DNA template having the target sequence, a heat-resistant DNA polymerase, typically Tag or Pfu, a pair of primers, i.e. a pair of short, single-stranded sequences complimentary to the 3′ end of the target sequence, and nucleotides to form the sequence copies.[0003]Once the solution is mixed in a PCR tube, the tube is placed in a thermal cycler and exposed to a series of temperature cycles which enable the target sequence to be denatured, each primer to anneal (...

AI Technical Summary

Benefits of technology

[0017]According to a first aspect of the present invention, there is provided a PCR fluorescence reference standard comprising 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. The main advantage of the standard provided is that it allows validation of PCR instruments which have, until now, not been validated. Moreover, the fluorophore suspended in thermoplastic polymer matrix is reusable and stable. In addition, the suspended fluorophore of the present invention can be used for all types of thermal cyclers, i.e. standard PCR and qPCR machines.

[0018]Advantageously, the polymer matrix comprises poly methyl methacrylate, hereinafter PMMA. The advantage of using PMMA is that it possesses good optical properties and it does not have absorbance peaks.

Problems solved by technology

This is useful, for example in forensic applications in which it is necessary to determine the quantity of starting material However, if the reaction is carried out until it reaches the plateau phase, it is not possible to quantify the amount of starting material.

There is currently no standard method used to validate thermal cyclers having means for measuring fluorescence of a sample; this is highly undesirable because it means that fluorescence measurements obtained in thermal cyclers are not traceable.

The main disadvantage of this device is that it can only be used to validate a fluorescence signal obtained in a standard fluorimeter and cannot be used to validate a fluorescence signal obtained in a thermal cycler.

Adapting this device for use in a thermal cycler, if at all possible, would be very difficult and expensive; in other words, adapting this device for use in thermal cyclers is unfeasible because of difficulty, expense and potential inadequacy of the adapted product.

Currently, the only way to validate a thermal cyder is the use of a liquid reference sample, which requires very accurate preparation and will not in any event make the measurements traceable due to the use of the liquid reference.

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