RT-PCR amplification

The object of the invention is an improved collection of fluorescent light from the sample during the RT-PCR amplification by the addition of a translucent layer in the optical path between the RT-PCR reaction mixture and the fluorescence detector and the preparation procedure of the mentioned translucent layer.

Described are a procedure and a substance, which form a transparent layer, which improves the sensitivity and repeatability of fluorescence measurements in the real-time polymerase chain reaction, in continuation RT-PCR. The invention improves the collection of fluorescent light from the sample during RT-PCR amplification. It acts on the principle of a substance, which is optically permeable,, and which is situated in the optical path between the reaction mixture and the fluorescence detector and at the same time doesn't mix with water. An example of such a substance is a paraffin or another mineral oil. The substance is added into the RT-PCR reaction mixture, where it enables a better collection of the dissipated fluorescent light and its directing into the detector of the RT-PCR cycler. The better collection is achieved by the effect of forming a lens above the reaction mixture and by preventing water evaporation from the reaction mixture and, consequentially, the condensation of the evaporated water onto the surfaces in the optical path, as for instance with a covering foil, which covers the microtiter plate, and therewith by preventing additional light dissipation from the reaction mixture. The substance is added before the start of the chain reaction, namely it can be added into the RT-PCR reaction mixture or directly into the sample. The substance significantly reduces the number of cycles, necessary for the achievement of the fluorescence detection threshold and thereby shortens the time, necessary for the acquirement of results, smaller is also the scatter of measurement results, and therewith a smaller number of repetitions or of parallel measurements of the same sample is necessary.

The object of the invention is therefore a method and a translucent layer, i.e., a substance for improving the fluorescence measurement results in the real-time polymerase chain reaction (RT-PCR). The addition of the substance into the RT- PCR reaction mixture or directly into the sample before the start of the chain reaction improves the fluorescence detection. The substance doesn't mix with water, it is optically permeable and is situated in the optical path between the reaction mixture and the fluorescence detector, and it acts as a lens between the sample and the fluorescence detector and prevents water evaporation from the reaction mixture. The substance is not toxic and doesn't inhibitory affect the chemical course of the reaction with the polymerase. An example of such a substance is a paraffin or another mineral oil or any other substance having the mentioned characteristics.

The detection of real-time nucleic acid amplification in RT-PCR is performed by the simultaneous fluorescence measurement of the formed products. Precisely the fluorescence measurement brings besides advantages also some challenges, since any light dissipation in the sample or in the optical path to the fluorescence detector reduces the sensitivity and the reliability of the measurement apparatus. Consequentially, for a reliable result it is necessary to prolong the amplification time by adding amplification cycles as well as to increase the number of necessary parallel measurements for the purpose of a better middle value estimation. Particularly demanding is the quantification of the target nucleotide sequences in the sample, which requires especially precise amplification. In the present state of the art there are no methods for improving the fluorescence measurement reliability in the real-time polymerase chain reaction by the addition of substances into the reaction mixture or into the sample.

The aim of the invention is the introduction of a method and a substance, which substantially improve the fluorescence detection in the real-time polymerase chain reaction and thereby the repeatability, sensitivity and reliability of the measurement results of the amplified target nucleotide sequences. The method statistically significantly increases the fluorescent signal from the sample and thereby apparently improves the reaction yield and reduces the measurement scatter of parallel measurements. The detection error is reduced and thus to a great extent merely the error remains, bound to the unreliability in the preparation of the appropriate quantity of the reaction mixture and of the sample. Higher repeatability and shortened reaction time enable smaller consumption of chemicals, which reduces the price of the procedure and reduces the environment loading.

Mineral oils, which are an example of the substance, were used in classical procedures for the polymerase chain reaction (PCR), where the detection of products doesn't occur in real time, for preventing the sample evaporation and thereby improving the reaction yield, described in patents US005411876A, US005576197A. In RT-PCR the mineral oils are not added, since the detection of the reaction products is completely different from the classical procedures.

DESCRIPTION OF THE INVENTION:

The invention is described by means of figures, representing:

Figure 1 : A comparison of RT-PCR results (amplification of the 99 kb DNA target using SybrGreen I as the fluorophore). Black curves present the detection of DNA amplification in the presence of the paraffin oil. Gray curves present the detection of an equal product without the paraffin oil. In the detection in the presence of the paraffin oil the increase of the fluorescent signal, coming to the detector in the RT- PCR cycler, is evident. Visible is a smaller variability between triplicates and a higher sensitivity.

Figures 2 a and b: A comparison of melting curves for the amplification results, represented in Figure 1. Black curves present melting curves in the presence of the paraffin oil. Gray curves present melting curves without the presence of the paraffin oil. Smaller variability between repetitions is evident when using the paraffin oil. The invention is based on the addition of a translucent layer, onto the surface of the RT-PCR reaction mixture, of a substance, which doesn't mix with water and is optically permeable, being situated in the optical path between the reaction mixture and the fluorescence detector, acting as a lens between the sample and the fluorescence detector and preventing water evaporation, into the reaction mixture or the sample before the start of the polymerase chain reaction. The substance can have the described characteristics already immediately at the addition or they express themselves, however, afterwards before or during the reaction by physical or chemical procedures. The substance forms a water- impermeable layer, which acts as the lens for the fluorescent light collection.

The substance enables better collection of the dissipated fluorescent light and its directing into the detector of the RT-PCR cycler. Better collection is achieved by the effect of forming a lens between the reaction mixture and the fluorescence detector and by preventing water evaporation from the reaction mixture and consequentially the condensation of evaporated water onto the surfaces in the optical axis, as for instance with a covering foil, which covers the microtiter plate, and therewith by preventing additional light dissipation from the reaction mixture - if the layer is deposited above the reaction mixture.

The substance can partly adhere to the microtiter plate well walls, but during the centrifugation or heating in the RT-PCR cycler, which are parts of the standard procedure in the nucleic acid amplification, it detaches and swims up to the surface, where it bends like a lens. The substance can be added in the final form or, however, in the form, which is transformed into the final form with all required characteristics by a physical or chemical transformation. An example: the paraffin oil can be added in the form of an opaque emulsion, which forms a translucent layer only after the centrifugation and heating.

The basic method of the sample preparation, of the reaction mixture preparation and of the addition of all necessary chemicals for RT-PCR is performed according to directions of the producers and is not changed by the invention, with the exception of the substance addition itself.

The improved collection of the fluorescent light from the sample during RT-PCR amplification according to the invention is therefore characterized in that a translucent layer is added in the optical path between the RT-PCR reaction mixture and the fluorescence detector, which collects the dissipated light and prevents evaporation and condensing of water in the optical axis, when the layer is deposited above the reaction mixture. The translucent layer is of a substance, which doesn't mix with water and is optically permeable, whereby the substance is situated in the optical path between the reaction mixture and the fluorescence detector and forms a translucent layer between the reaction mixture with the sample and the fluorescence detector and can prevent water evaporation from the reaction mixture. The substance is not toxic and doesn't inhibitory affect the chemical course of the reaction with the polymerase. The substance can have the described characteristics already immediately at the addition or they express themselves, however, afterwards, before or during the reaction by physical or chemical procedures. The substance can be a material, having the described characteristics, when added into the reaction mixture, into the sample or directly into the microtiter plate well before the start of the polymerase chain reaction.

The substance can be any compound, being in the liquid aggregate state and translucent in the temperature range from 20 to 100 °C. The substance has to form a translucent layer between the reaction mixture and the fluorescence detector. With regard to the detector position in the measuring apparatus the substance has to be lighter or heavier than the reaction mixture. It means if the detector is above the sample, the substance has to be translucent, when it is lighter than water (density 0.8 - 1 g/cm ³ ). If the detector is below the sample, the substance has to be translucent, when it is heavier than water (density 1.01 - 1.1 g/cm ³ ). Such substances are: all mineral oils, e.g. a paraffin oil, waxes, organic oils, silicon oils and lipids. The preparation method of RT-PCR with the added translucent layer in the optical path between the reaction mixture and the fluorescence detector is characterized in that into the reaction mixture or into the sample itself before the reaction between 5 and 200 microliters of the described substance are added, such that it completely fills up the surface of the water solution of the reaction mixture and forms a lens. In case the reaction is performed in the microtlter plate wells, usually between 5 and 20 microliters of the described substance are added. It can be added together with the sample, previously into the reaction mixture or into the mixture in the well or in the test tube.

EXAMPLE OF APPLICATION

As the substance mineral oils can be used, such as a paraffin oil. Into the reaction mixture or into the sample itself, before the reaction, between 5 and 200 microliters of the paraffin oil are added - so that it completely fills up the surface of the water solution of the reaction mixture and forms the lens. In case the reaction is performed in the microtiter plate wells, usually between 5 and 20 microlitres of the oil are added. The oil can be added together with the sample, previously into the reaction mixture or into the mixture in the well or in the test tube.