CN117660165A - Multi-point detection ultra-multiple digital PCR optical path reading module and reading method - Google Patents

Abstract

The invention provides a multi-point detection ultra-multiple digital PCR optical path reading module and a reading method, wherein the optical path reading module adopts a multi-point detection mode, so that signal crosstalk among multiple channels is well reduced, and the signal resolution of a single channel is improved. The single reading device adopts the principles of laser beam combination and fluorescence beam splitting, so that the volume of an instrument light path part is greatly reduced, and the component cost of an optical part is reduced. The reading method integrates, counts and eliminates the detection signals among multiple points, reduces the problem of crosstalk of fluorescent signals emitted among multiple channels, realizes a PCR reading system with more than 6 channels, realizes the grading capability of fluorescent signals with different dye concentrations of 6-10 orders in a single channel, and meets the reading requirement of the ultra-multiple PCR reagent.

Description

Multi-point detection ultra-multiple digital PCR optical path reading module and reading method

Technical Field

The invention relates to the technical field of digital PCR, in particular to a reading module and a reading method for a multi-point-position detection ultra-multiple digital PCR optical path.

Background

PCR (Polymerase Chain Reaction ) is a molecular biological technique for amplifying specific DNA fragments, and has undergone three generations of development. The first generation of PCR, namely ordinary PCR, is a qualitative result obtained by gel electrophoresis, and is an end-point PCR technique. The second generation PCR, namely fluorescent quantitative PCR, uses fluorescent probe to trace the PCR product, monitors the amplifying process in real time, and determines the nucleic acid amount by means of standard curve or reference gene, thus realizing relative quantification and having very wide application. The third generation PCR, namely digital PCR, can determine the absolute number of target molecules to be detected as low as a single copy by carrying out micro unitization treatment on a sample, and is an absolute quantitative PCR technology. The microdroplet digital PCR is one kind of digital PCR technology, and is different from traditional PCR technology, and it includes the first microdroplet treatment of sample before amplification, the subsequent PCR amplification, the detection and analysis of fluorescent signal of each microdroplet, and finally the initial copy number or concentration of target molecule based on Poisson distribution principle and the number and proportion of positive microdroplets. In the existing microdroplet digital PCR, the micro-fluidic technology is used to make the sample into microdroplets before the DNA amplification. Such that each droplet contains only a single or no target nucleic acid molecule. After PCR amplification, each droplet is subjected to fluorescent signal detection, the droplets detected with strong fluorescent signals are labeled as positive droplets, and the droplets with weak or no fluorescent signals are labeled as negative droplets. The poisson distribution principle requires that positive droplets and negative droplets are counted simultaneously, and the detection of the negative droplets is judged by weak fluorescence emitted by fluorescent probes which are not involved in the reaction in the droplets, however, the following problems exist: the very weak fluorescent signal is easily interfered by environmental noise, and may be misinterpreted as noise, resulting in a small number of detected negative droplets, such that the number of droplet samples cannot be counted accurately.

The PCR reader on the market at present mainly comprises two technical routes, namely an integral photographing type technical route, and the technical route is the most common reader with 6 channels on the market at present, but the photographing type reader has the problems of poor fluorescence signal grading capability of different concentrations and stronger signal interference between liquid drops, and cannot meet the reading requirement of the ultra-multiple PCR reagent.

Still another is the flow-type technology route, this technology route is the most common reading instrument with 4-6 channels on the market at present, but the fluorescence signal of single channel to different dye concentration is weak to the ability of classifying, generally only can divide to 3 to 4 order. Nor can it meet the reading requirements of our super multiplex PCR reagents.

Disclosure of Invention

The invention aims to solve the problems of the prior art, and provides a multi-point detection ultra-multiple digital PCR optical path reading module and a reading method, which integrate, count and reject detection signals among multiple points, reduce the problem of crosstalk of fluorescent signals emitted among multiple channels, realize a PCR reading system with more than 6 channels, realize the grading capability of fluorescent signals with different dye concentrations of 6-10 orders in a single channel, and meet the reading requirement of ultra-multiple PCR reagents.

The invention provides a multi-point detection ultra-multiple digital PCR optical path reading module, which comprises optical path reading devices distributed along a plurality of points on two sides of a detection flow channel pool, wherein the optical path reading devices comprise a light source, a beam combining device, a focusing lens, a light splitting device and PMT fluorescent receivers, the light source is a plurality of groups of lasers emitting different wavelengths, the lasers emitted by the lasers are combined by the beam combining device and then irradiate onto one point in the detection flow channel pool through the focusing lens, and fluorescence emitted by liquid drops in the detection flow channel pool is collected through the focusing lens and then reflected into different PMT fluorescent receivers through the light splitting device to be received.

Further improved, the beam combining device and the beam splitting device both adopt dichroic mirrors.

Further improved, the beam combining device is a beam combining dichroic mirror arranged in front of the emitting end of each laser, each beam combining dichroic mirror reflects the laser emitted by the corresponding laser and transmits the laser of other wave bands passing through.

The light splitting device comprises a main light splitting dichroic mirror and a plurality of secondary light splitting dichroic mirrors, wherein the main light splitting dichroic mirror is arranged at the front end of the focusing lens, and the main light splitting dichroic mirror transmits the laser after beam combination and reflects the fluorescence returned by beam combination; the plurality of times of dichroic mirrors are arranged at the front end of each PMT fluorescence receiver, so that the PMT fluorescence receiver receives fluorescence in a corresponding wave band.

The invention also provides a multi-point-position-detection super-multiple digital PCR reading method, which utilizes a multi-point-position-detection super-multiple digital PCR optical path reading module to detect liquid drops passing through a detection flow channel pool and integrate detected signals, eliminates invalid liquid drops, and then carries out signal classification according to the signal amplitude of all valid liquid drops.

The specific reading method is as follows:

1) The detection liquid drops with fluorescent dye sequentially pass through the detection flow channel pool, the passing liquid drops are detected by using the ultra-multiple digital PCR optical path reading module for multi-point detection, and the principles of laser beam combination and fluorescent light splitting are adopted, so that the volume of an optical path part of the instrument is greatly reduced, and the component cost of an optical part is reduced.

2) Counting pulse width and amplitude signals of all liquid drops, integrating all liquid drop signals measured among all the points, and ensuring that signals of each liquid drop passing through all the detection points can be integrated and corresponding; when the drop passes through the first point, the next start time t is recorded, the time for reaching each point later should be t+nΔt, and assuming that the distance between each point is L and the drop flow rate is V, the time Δt=l/V for the drop from one detection point to the next detection point.

3) And removing all the liquid drops according to the pulse widths, firstly collecting waveform signals of all the liquid drops, then primarily rounding and counting the pulse widths of all the liquid drops, and removing the liquid drop signals exceeding 30% of the mode pulse widths on the basis of primarily counting the mode pulse widths.

4) Signal classification is performed according to the signal amplitude of all the effective droplets.

The invention also provides a multi-point-position-detection ultra-multiple digital PCR reader, which comprises a detection flow channel pool, a liquid channel storage area, a liquid channel pump valve module and a mechanical transmission module; the mechanical transmission module comprises a triaxial mechanical transmission mechanism and a liquid suction needle, the triaxial mechanical transmission mechanism controls the liquid suction needle to suck samples of different hole sites on the pore plate, the liquid suction needle forms a detection liquid path between the liquid path pump valve module and the detection flow path pool and liquid path storage area, and the two sides of the detection flow path pool are distributed with ultra-multiple digital PCR light path reading modules for multi-point detection. The detection flow passage pool is connected with a three-way valve, and a cleaning liquid path is formed among the detection flow passage pool, the liquid path storage area and the liquid suction needle head through the three-way valve and the liquid path pump valve module.

The invention has the beneficial effects that:

1. through the optical reading module, the light source and the dye are matched and combined according to the design rule and then are divided into a plurality of groups, a plurality of point positions at different distances on the flow channel pool are detected, and a multi-point detection mode is adopted, so that signal crosstalk among multiple channels is well reduced, and the signal resolution of a single channel is improved.

2. The single reading device adopts the principles of laser beam combination and fluorescence beam splitting, so that the volume of an instrument light path part is greatly reduced, and the component cost of an optical part is reduced.

3. Through the designed algorithm, detection signals among multiple points are integrated, counted and removed, the problem of crosstalk of fluorescent signals emitted among multiple channels is reduced, a PCR reading system with more than 6 channels is realized, the grading capability of fluorescent signals with different dye concentrations of 6-10 orders is realized through a single channel, and the reading requirement of the ultra-multiple PCR reagent is met.

4. A special PCR reader is designed, and is matched with an optical reading module and a PCR reading method, so that continuous detection of a plurality of samples on the pore plate is realized.

5. The PCR reader is provided with a cleaning liquid path, and is convenient to clean before each reading or under the condition of long-time non-use.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the overall layout of a PCR reader;

FIG. 2 is a schematic diagram of a single-point reading light path and a partial flow channel pool of a PCR reader;

FIG. 3 is a schematic view of a pipette tip;

fig. 4 is a schematic diagram of an optical path reading module.

In the figure, 1 is a clean fluorine oil storage area, 2 is a waste oil barrel, 3 is a reading light path, 4 is a first three-way valve, 5 is a second three-way valve, 6 is a detection flow passage pool, 7, 8, 9 and 10 are first to fourth micropumps, 11 is a triaxial mechanical transmission mechanism, and 12 is a liquid suction needle.

A1-A3 are first to third lasers, A4-A6 are first to third photomultiplier tubes, A7, A8 and A9 are first to third beam-combining dichroic mirrors, A10, A11 and A12 are first to third beam-splitting dichroic mirrors, A13 is a main beam-splitting dichroic mirror, A14 is a focusing lens and C1 is a detection point.

3-1 is a sample suction port, and 3-2 is a carrier oil inlet.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The invention provides a multi-point-position-detection ultra-multiple digital PCR reader, which is particularly shown in fig. 1, and comprises an optical path reading module, a liquid path storage area, a liquid path pump valve module and a mechanical transmission module, wherein the optical path reading module is shown in fig. 4.

The liquid path storage area comprises a clean fluorine oil storage area 1 and a waste oil barrel 2, the fluorine oil is mainly used for lavaging the pipeline and used as carrying oil when testing liquid drops, and the waste oil barrel 2 is used for storing cleaned waste oil and tested liquid drops.

3 are the reading light paths of a plurality of points, the detection points are different from 2 to 6, the design of the reading light path on each point is different, the main differences are different in the wave bands of the lasers, and the corresponding detectors, optical filters and the like are different. And the number of channels combined is different. The method comprises the steps of firstly reasonably selecting a laser excitation wave band and a detector receiving wave band according to a rule that excitation and receiving are not interfered and a principle that each channel is not interfered. And then reasonably grouping and combining the channels. So the reading light path of one detection point is generally obtained by combining 1 to 5 channels in unequal mode. Therefore, the whole multi-point reading module has different reading light paths of each detection point.

Fig. 2 shows a schematic diagram of a single point reading light path and a flow channel pool, the whole light path design adopts the ideas of beam combination and beam splitting, light beams of three lasers are firstly combined through a first beam combination dichroic mirror to a third beam combination dichroic mirror, the three laser beams are irradiated on the same point C1 through a focusing lens A14, mixed color fluorescence excited by the point is also collected through the focusing lens A14, then is reflected to a receiving light path through a main beam splitting dichroic mirror, the mixed color fluorescence is split through the first beam combination dichroic mirror to the third beam splitting dichroic mirror, a third beam splitting dichroic mirror A10 penetrates through a fluorescence wave band corresponding to a third photomultiplier A6, other wave bands are reflected, the reflected fluorescence passes through a second beam splitting dichroic mirror A11, and the fluorescence corresponding to a second photomultiplier A5 is reflected through other wave bands. The transmitted fluorescence is irradiated to the first dichroic mirror a10, and the first dichroic mirror a10 may be a simple mirror, and reflects the fluorescence to the first photomultiplier tube A4. All fluorescence is finally collected to the corresponding first to third photomultiplier tubes (PMT fluorescence receivers).

The length of the detection flow channel pool 1-6 is about 10 to 20mm, and 2 to 6 detection points are distributed on the whole detection flow channel from right to left. Each detection point is different in distance from 1 to 10mm according to parameters of the reading light path lens, and the distance between the detection points is as small as possible, so that the pulse width stability of the same liquid drop signal passing through each point is improved.

In addition, the multi-point reading module can completely work only by combining a special algorithm which is designed independently, and the specific algorithm is as follows:

the whole algorithm idea of the liquid drop signal reading is that firstly, pulse width and amplitude signals of all liquid drops are counted, all liquid drop signals measured among all the point positions are integrated, and the fact that signals of all liquid drops passing through all the detection point positions can be integrated and corresponding is guaranteed. And then all the liquid drops are removed according to the pulse width, and invalid liquid drops are removed, so that the accuracy of the test result of the user can be improved. And finally, signal classification is carried out according to the signal amplitude values of all the effective liquid drops.

1. Pulse width rejection:

firstly, waveform signals of all liquid drops are collected, then pulse widths of all liquid drops are counted, when the pulse widths are counted preliminarily, only integer bits are taken, so that the mode signal width of all liquid drops, which appears in the pulse widths, can be found, then all liquid drop pulse widths are removed on the basis of the mode pulse width, and only the liquid drop signals with the pulse widths exceeding 30% of the mode pulse width are removed, because the pulse widths of all liquid drops are considered to be within 30% of the mode pulse width under normal conditions. If it exceeds the specification that this is an abnormal drop. It is not significant for our detection.

2. Counting the number of liquid drops: the method for counting the number of the liquid drops is to collect all pulse signals by taking a first detection point as a standard and then count the liquid drops.

3. Droplet signal integration:

the signals generated by exciting the liquid drops by different lasers at each point are integrated, namely the liquid drops are in one-to-one correspondence with each signal, so that the next signal elimination and signal arrangement can be performed, and the final detection result is output. The method is that when the liquid drop passes through the first point, the next starting time t is recorded, the time for reaching each point later is t+nDeltat, assuming that the distance between each point is L, the liquid drop flow rate is V, and then the time for reaching the next point from one detection point is Deltat=L/V. In practice the distance design between the various spots of our reader is different, as better avoidance of signal crosstalk between systems is considered. All droplets are subjected to signal correspondence according to the method.

The first three-way valve 4 is provided with an oil inlet path at two sides and a sample inlet path in the middle. The second three-way valve 5 divides one way of oil into two ways.

Four micropumps control the liquid path of the whole reading instrument. The fourth micro-pump 10 is a cleaning liquid path for sucking fluorine oil, the third micro-pump 9 is a cleaning liquid path for carrying oil and liquid inlet of samples, the second micro-pump 8 is a cleaning needle head and needle head liquid inlet, and the first micro-pump 7 is a pumping pump and liquid suction of waste liquid.

The whole liquid path working principle is specifically described as follows: the liquid path is cleaned before each reading, and lavage is performed under the condition of long-term non-use. The liquid path cleaning step is as follows: firstly, the second to fourth micropump extracts the fluorine oil from the fluorine oil barrel, then the fluorine oil is pushed out at a higher speed, and the fourth micropump 10 mainly cleans the sample injection liquid path of the runner pool and the inner hole of the liquid suction needle. The third micropump 9 mainly cleans the flow channel pool carrier oil inlet liquid path. The second micropump 8 mainly cleans the outer hole of the pipette tip. In the cleaning process, waste liquid in the flow channel pool is mainly sucked into the waste liquid barrel by the first micro pump 7, and waste oil generated by the liquid suction pinhole is also sucked into the liquid waste barrel by the first micro pump 7. If the liquid path system is left for a long time, the same cleaning process is carried out for 3 times before use, so that the whole liquid path system is ensured to be cleaned.

The whole set of triaxial mechanical transmission mechanism 11 is responsible for moving the pinhead to samples of different hole sites on the 96-well plate.

A pipette tip 12 for pipetting samples from within the 96 well plate.

The structure of the liquid suction needle is shown in the following figure 3, and comprises a sample suction port 3-1 and a carrier oil inlet 3-2, and the suction port 3-1 and the carrier oil inlet 3-2 are simultaneously used for feeding oil for cleaning during cleaning. When the sample is sucked, the oil carrying inlet 3-2 firstly injects a certain amount of oil from the outer hole into the sample hole, and then the sample sucking port 3-1 is used for sucking again, so that the sample is completely sucked. During sample sucking, the outer hole of the needle head does not enter the sample.

In this specification, each embodiment is described in a progressive manner, and identical and similar parts of each embodiment are all referred to each other, and each embodiment mainly describes differences from other embodiments. In particular, for the equipment examples, what has been described above is merely a preferred embodiment of the invention, which, since it is substantially similar to the method examples, is described relatively simply, as relevant to the description of the method examples. The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, since modifications and substitutions will be readily made by those skilled in the art without departing from the spirit of the invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims (10)

1. The utility model provides a multiple digital PCR light path reading module of super that multiposition detected which characterized in that: the device comprises an optical path reading device distributed along a plurality of points on two sides of a detection flow channel pool, wherein the optical path reading device comprises a light source, a beam combining device, a focusing lens, a light splitting device and a PMT fluorescent receiver, wherein the light source is a plurality of groups of lasers emitting different wavelengths, laser emitted by the lasers irradiates one point in the detection flow channel pool through the focusing lens after being combined by the beam combining device, and fluorescent light emitted by the liquid drops in the detection flow channel pool is collected through the focusing lens and then reflected to different PMT fluorescent receivers through the light splitting device to be received.

2. The multi-spot detection ultra-multiple digital PCR optical path reading module of claim 1, wherein: the beam combining device and the beam splitting device both adopt dichroic mirrors.

3. The multi-spot detection ultra-multiple digital PCR optical path reading module of claim 2, wherein: the beam combining device is a beam combining dichroic mirror arranged in front of the emitting end of each laser, each beam combining dichroic mirror reflects laser emitted by the corresponding laser and transmits laser of other wave bands passing through.

4. A multiplex digital PCR optical path reading module for multi-point detection according to claim 2 or 3, wherein: the light splitting device comprises a main light splitting dichroic mirror and a plurality of secondary light splitting dichroic mirrors, wherein the main light splitting dichroic mirror is arranged at the front end of the focusing lens, and the main light splitting dichroic mirror transmits the laser after beam combination and reflects the fluorescence returned by the beam combination; the plurality of times of dichroic mirrors are arranged at the front end of each PMT fluorescence receiver, so that the PMT fluorescence receiver receives fluorescence in a corresponding wave band.

5. A multiplex digital PCR reading method for multi-point detection is characterized in that: the method comprises the steps of detecting liquid drops passing through a detection flow channel pool by utilizing a multi-point detection ultra-multiple digital PCR optical path reading module, integrating detected signals, removing invalid liquid drops, and carrying out signal classification according to signal amplitudes of all valid liquid drops.

6. The method for reading the ultra-multiple digital PCR for multi-point detection according to claim 5, wherein the specific reading method is as follows:

1) Enabling the detection liquid drops with fluorescent dye to sequentially pass through a detection flow channel pool, and detecting the passed liquid drops by using a multi-point detection ultra-multiple digital PCR optical path reading module;

2) Counting pulse width and amplitude signals of all liquid drops, integrating all liquid drop signals measured among all the points, and ensuring that signals of each liquid drop passing through all the detection points can be integrated and corresponding;

3) All the liquid drops are removed according to the pulse width, and invalid liquid drops are removed;

4) Signal classification is performed according to the signal amplitude of all the effective droplets.

7. The method for multiplex digital PCR reading for multi-spot detection as in claim 6 wherein: the specific process of the droplet signal integration in the step 2) is as follows: when the drop passes through the first point, the next start time t is recorded, the time for reaching each point later should be t+nΔt, and assuming that the distance between each point is L and the drop flow rate is V, the time Δt=l/V for the drop from one detection point to the next detection point.

8. The multi-spot detection ultra-multiple digital PCR optical path reading module of claim 6, wherein: the invalid liquid drop removing process in the step 3) specifically comprises the following steps: firstly, collecting waveform signals of all liquid drops, then primarily rounding and counting pulse widths of all liquid drops, and eliminating the liquid drop signals exceeding 30% of the mode pulse width on the basis of the primarily counted mode pulse width.

9. A super multiple digital PCR reader for multi-point detection is characterized in that: the device comprises a detection flow passage pool, a liquid path storage area, a liquid path pump valve module and a mechanical transmission module; the mechanical transmission module comprises a triaxial mechanical transmission mechanism and a liquid suction needle, the triaxial mechanical transmission mechanism controls the liquid suction needle to suck samples of different hole sites on the pore plate, the liquid suction needle forms a detection liquid path between the liquid path pump valve module and the detection flow path pool and the liquid path storage area, and the ultra-multiple digital PCR optical path reading module for multi-point detection as claimed in claim 1 is distributed on two sides of the detection flow path pool.

10. The multiple spot detection ultra-multiplex digital PCR reader as in claim 9 wherein: the detection flow passage pool is connected with a three-way valve, and a cleaning liquid path is formed among the detection flow passage pool, the liquid path storage area and the liquid suction needle head through the three-way valve and the liquid path pump valve module.