RU2774888C1 - Apparatus for recording pcr results with a monochromator - Google Patents

Abstract

FIELD: computing technology.

SUBSTANCE: invention relates to an apparatus for recording the results of a polymerase chain reaction (PCR). Apparatus for recording PCR results comprises a light source (1) with a wide emission spectrum, a lighting lens (8), a cell (9) with samples, and a recording system. A monochromator is installed between the light source (1) and the lighting lens (8), providing illumination of the samples with monochromatic excitation light, as well as return of fluorescent emission in the course of autocollimation by the lens (8) to the output slot (7) of the monochromator and focusing thereof into the slot (10) located in the plane of the input slot (3) of the monochromator and distanced therefrom by the value of linear dispersion of the monochromator, corresponding to the difference in the wavelengths of the exciting and fluorescent emission.

EFFECT: apparatus allows for PCR diagnostics using stains with different absorption and fluorescence spectra, and provides a possibility of resetting the wavelength of exciting emission without the need for application of narrow-band filters, temporary degradation whereof can lead to errors in the operation of the apparatus.

1 cl, 2 dwg

Description

Technical field

The invention relates to registration devices. In particular, the invention relates to a device for recording PCR results using dyes with different absorption and fluorescence spectra. The device can be used in medicine, control of microbiological contamination of the environment and food, forensics.

State of the art

The polymerase chain reaction is one of the methods of molecular biology. This method is based on repeated copying (amplification) of certain sections of DNA and RNA in the process of repeated temperature cycles.

On FIG. Figure 2 shows a generalized diagram of the optical-electronic unit of the device intended for conducting and recording PCR results. The reaction mixture (14), which contains the studied biological material, is placed in a container (15), which, in turn, is located in an amplifier (16), which performs cyclic temperature exposure to material samples. As a result of the polymerase chain reaction, in the presence of fragments of the desired DNA in the sample, an exponential increase in their number occurs. This increase is recorded by the fluorescent radiation of the dye, which is introduced into the reaction mixture before diagnosis. The intensity of fluorescent radiation increases in proportion to the amount of amplification product. For excitation of fluorescent radiation and its registration, an optoelectronic unit (17) is used, which includes an objective (18), a source of quasi-monochromatic exciting radiation (19), a radiation receiver (20), a spectrum splitter (21), an excitation filter (22) and registration filter (23), which are narrow-band interference filters. The fluorescence spectra of dyes are shifted to the long wavelength region relative to the spectra of exciting radiation by 30–40 nm. Therefore, the passband of the filter (23) is shifted relative to the passband of the filter (21) by the same amount.

Among the devices, the optical-electronic unit of which is built according to the above scheme, one can note a device for PCR diagnostics (CFX96 Touch Real-Time PCR Detection System from Bio-Rad [1]), a device for PCR diagnostics (amplifier detecting DTprime 4M1 from DNA-Technology [2]). In these devices, the sources of exciting radiation are light-emitting diodes.

The closest in technical essence to the proposed invention is a real-time PCR diagnostic device (7500 Fast Dx Real-Time PCR System from Thermo Fisher Scientific [3]). The device contains a cuvette for samples, a thermal cycler, and an optoelectronic unit consisting of a fluorescence excitation source based on a halogen lamp and a recorder based on a camera with a CCD array. The device has the ability to conduct multiplex studies at five fixed wavelengths of exciting radiation while using up to five dyes. To do this, the device provides for the installation of replaceable excitation and registration filters.

The essence of the invention

The objective of the invention is to ensure the operation of the device with radiation of any wavelength.

The essence of the proposed invention lies in the fact that the selection of monochromatic radiation of the required wavelength for excitation of fluorescence is carried out by a monochromator. This monochromator also selects fluorescent radiation from the studied samples and directs it to the entrance pupil of the registration system.

The advantage of the proposed invention in comparison with the prototype lies in the possibility of tuning the wavelength of the exciting radiation and the absence of the need to use narrow-band filters, the temporal degradation of which can lead to errors in the operation of the device.

Brief description of the drawings

Fig. 1 is a diagram of the optical-electronic unit of the present invention.

Fig. 2 - a generalized diagram of the optical-electronic unit of the device for PCR diagnostics.

Implementation of the invention

On FIG. 1 shows an example of a specific design of the optical-electronic unit of the device for PCR control with a monochromator.

Light from a radiation source (1) with a wide spectrum, for example, from a tungsten halogen lamp, is focused by a condenser (2) on the entrance slit (3) of the monochromator. The monochromator consists of input (4) and output (5) lenses, a dispersive element (6), for example, a prism of constant Abbe deflection angle. When the exit slit (7) of the monochromator is located in the front focal plane of the illumination objective (8), the samples in the cuvette (9) are illuminated by a parallel beam of monochromatic light, the wavelength of which can be changed by turning the prism (6). The fluorescent radiation of the samples in the autocollimation course is returned by the lens (8) to the slit (7) and, in the reverse course of the rays, is focused into the slit (10), which is in the plane of the entrance slit (3) and is separated from it by the value of the linear dispersion of the monochromator corresponding to the difference in wavelengths exciting and fluorescent radiation and a component of 30-40 nm. After passing through the prism (11), the fluorescent radiation is collected by the lens (12), in the image plane of which there is a photosensitive layer of the radiation receiver (13), for example, a CCD array.

List of references to the application

Device for recording PCR results with a monochromator

1. BioRad. Real-Time PCR Systems [Electronic resource]. URL: https://www.bio-rad.com/ru-ru/product/cfx96-touch-real-time-pcr-detection-system (Accessed: 04/01/2020).

2. DNA Technology. Detecting amplifiers [Electronic resource]. URL: https://www.dna-technology.ru/dnaproducts/equipments/rtamplificators/dt_prime (date of access: 04/01/2020).

3. Thermo Fisher Scientific. Real Time PCR. Applied Biosystems Real-Time PCR Instruments [Electronic resource]. URL: https://www.thermofisher.com/en/en/home/life-science/pcr/real-time-pcr/real-time-pcr-instruments/7500-fast-real-time-pcr-system. html (date of access: 04/01/2020).

Claims (1)

Device for recording PCR results with a monochromator, containing a light source (1) with a wide spectrum of radiation, an illuminating lens (8), a cuvette (9) with samples and a registration system, characterized in that between the light source (1) and the illuminating lens (8) a monochromator is installed, which provides illumination of the samples with monochromatic excitation light, as well as the return of fluorescent radiation in the autocollimation course by the objective (8) to the exit slit (7) of the monochromator and its focusing into the slit (10), located in the plane of the entrance slit (3) of the monochromator and spaced from it by the value of the linear dispersion of the monochromator corresponding to the difference between the wavelengths of the exciting and fluorescent radiation.

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