Portable rapid analyzer for wheat molecular marker detection

Abstract

The invention provides a portable rapid analyzer for wheat molecular marker detection, which comprises an instrument box body, moving wheels, a wheat sample crushing cabin, a nucleic acid extraction module, a microfluidic amplification detection module, a carrying handle, a detection and analysis all-in-one machine, a result meter outlet, a sample placing box, a storage battery, a sampling power pump assembly, an ultrasonic generator and a data printer, the moving wheels are respectively connected to four corners of the lower part of the instrument box body through bolts; the moving wheels specifically adopt mute rubber universal wheels with brake pads; the wheat sample crushing cabin is integrally arranged at the lower part of the instrument box body. According to the invention, the whole process from a sample to a result can be completed within 25 minutes (ultrasonic disruption is carried out for 30 seconds, magnetic bead extraction is carried out for 5 minutes, rapid PCR is carried out for 15 minutes, and fluorescence detection is carried out for 5 minutes); the method is especially suitable for field real-time decision making, such as wheat variety authenticity identification, disease-resistant gene screening and other scenes with high timeliness requirements.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural biotechnology detection, and particularly relates to a portable rapid analyzer for wheat molecular marker detection. Background Art

[0002] Wheat is the food crop with the widest planting range, the widest consumption area, and the greatest impact on the three rural issues in China. China is the largest wheat producer and consumer in the world and has a decisive influence on the international wheat market. With the in-depth development of wheat distant hybridization and chromosome engineering research, exogenous chromosomes or chromosome fragments containing excellent genes such as disease resistance and stress resistance have been introduced into wheat, creating a large number of excellent intermediate materials, providing an important material basis for enriching the genetic basis of common wheat and improving traits. Molecular marker technology is a technology that uses the specificity at the molecular level of biology to detect and analyze the differences in individual DNA with molecular marker technology. According to different detection technologies, molecular marker technology can be divided into PCR (Polymerase Chain Reaction) technology, RFLP (Restriction Fragment Length Polymorphism) technology, SSR (Simple Sequence Repeat) technology, SNP (Single Nucleotide Polymorphism) technology, etc.

[0003] Molecular marker technology plays an irreplaceable and important role in wheat breeding, and can select wheat varieties suitable for different needs faster and more accurately, enriching people's taste and life. In addition, the Chinese Patent Publication No. is CN, and the invention creation name is SNP molecular marker for identifying common wheat varieties and SNP molecular marker detection method. However, the existing wheat molecular marker detection relies on laboratory PCR instruments and electrophoresis equipment, with problems such as large equipment volume, high cost, long detection cycle, or low detection accuracy of existing portable devices (such as constant temperature amplification detectors).

[0004] In view of this, it is very necessary to invent a portable rapid analyzer for wheat molecular marker detection. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a portable rapid analyzer for wheat molecular marker detection, so as to solve the problems that the existing wheat molecular marker detection relies on laboratory PCR instruments and electrophoresis equipment, with large equipment volume, high cost, long detection cycle, or the existing portable equipment (such as constant temperature amplification detector) has low detection accuracy. A portable rapid analyzer for wheat molecular marker detection includes an instrument box body, moving wheels, a wheat sample crushing chamber, a nucleic acid extraction module, a microfluidic amplification detection module, a hand-held handle, a detection and analysis integrated machine, a result output port, a sample placement box, a storage battery, a sampling power pump assembly, an ultrasonic generator and a data printer. The moving wheels are respectively bolted to the four corners of the lower part of the instrument box body; the moving wheels are specifically silent rubber universal wheels with brake pads; the wheat sample crushing chamber is integrally arranged at the lower part of the instrument box body; the nucleic acid extraction module and the microfluidic amplification detection module are screwed to the upper side of the front part of the instrument box body from bottom to top; the hand-held handle is screwed to the middle position at the rear side of the upper part of the instrument box body; the detection and analysis integrated machine is screwed to the upper side of the front part of the instrument box body; the result output port is screwed to the middle position at the upper right side of the instrument box body; the sample placement box is inserted into the wheat sample crushing chamber and is located at the lower side of the instrument box body; the storage battery is clamped at the bottom position inside the instrument box body; the sampling power pump assembly is screwed to the inside of the instrument box body; the ultrasonic generator is screwed to the left and right sides of the lower part of the instrument box body; the data printer is screwed to the upper right side inside the instrument box body.

[0006] Preferably, the sample placement box includes a longitudinal partition board, a transverse partition board, a mechanical crushing chamber, an ultrasonic crushing chamber and a hand-pulling handle. The longitudinal partition board and the transverse partition board are cross-connected and clamped inside the sample placement box; the front side of the sample placement box is divided into a mechanical crushing chamber; the rear side of the sample placement box is divided into an ultrasonic crushing chamber; the hand-pulling handle is screwed to the middle position at the upper side of the front part of the sample placement box.

[0007] Preferably, the sampling power pump assembly includes a suction pump, a connecting conduit, a left conduit, a right conduit, a telescopic device and a detection probe. The connecting conduit is threadedly connected to the inlet end of the suction pump; the left conduit and the right conduit are respectively threadedly connected to the left and right sides of the lower part of the connecting conduit; the output ends of the telescopic device are adhesively arranged with the lower parts of the left conduit and the right conduit respectively; the detection probes are respectively threadedly connected to the lower ends of the left conduit and the right conduit.

[0008] Preferably, the sample placement box is inserted into the inside of the wheat sample crushing chamber, and the ultrasonic generator is located on both sides of the ultrasonic crushing chamber.

[0009] Preferably, the data printer and the result output port are located on the same horizontal line and are interconnected.

[0010] Preferably, the suction pump and the telescopic device are respectively connected to the inner wall of the instrument box by screws.

[0011] Preferably, ventilation holes are provided on upper and lower sides of the front of the left-side conduit and the right-side conduit.

[0012] Preferably, the air outlet end of the suction pump is sleeved with a connecting pipeline, and the connecting pipeline is respectively connected to the nucleic acid extraction module, the microfluidic amplification detection module and the detection and analysis integrated machine.

[0013] Preferably, the microfluidic amplification detection module comprises a freeze-dried reagent chamber preloaded with DNA polymerase and dNTPs.

[0014] Preferably, the ultrasonic generator integrates ultrasonic fragmentation (frequency 40kHz±5%).

[0015] Preferably, the integrated detection and analysis machine adopts a replaceable chip design (the chip contains pre-loaded primers and the storage temperature is 4-25°C), and based on semiconductor temperature control (heating rate 10°C / s), 40 cycles of amplification can be completed in 15 minutes.

[0016] Preferably, the nucleic acid extraction module and the microfluidic amplification detection module have a built-in wheat variety database (containing ≥100 known variety marker features).

[0017] Preferably, the nucleic acid extraction module has a built-in enzyme catalytic reaction reagent.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The portable rapid analyzer for wheat molecular marker detection has the advantages of 1. significantly improved detection efficiency. Traditional laboratory detection requires 4 to 6 hours (DNA extraction 1 hour + PCR amplification 2 hours + electrophoresis analysis 1 hour), while the present invention can complete the entire process from sample to result within 25 minutes (ultrasonic disruption 30 seconds + magnetic bead extraction 5 minutes + rapid PCR 15 minutes + fluorescence detection 5 minutes); it is particularly suitable for real-time decision-making in the field, such as wheat variety authenticity identification, disease resistance gene screening and other scenarios with high timeliness requirements.

[0019] 2. Increased throughput allows simultaneous detection of 3 to 6 molecular markers (such as quality-related HMW-GS genes, disease resistance gene Fhb1, ergot resistance loci, etc.), while traditional equipment usually only supports single marker detection. Microfluidic chips support batch loading, and can process 8 to 12 samples in a single run (multi-chip slot version is required).

[0020] 3. "One-key start" design: Users only need to add samples, and subsequent DNA extraction, amplification, and detection are all automatically completed by the device without manual intervention. Intelligent result interpretation: The built-in algorithm automatically compares the wheat variety database and directly outputs "variety name + marker genotype" (such as "Zhengmai 366, Fhb1 resistant type RR"), eliminating the need for professionals to analyze electrophoresis bands or fluorescence curves.

[0021] 4. Reduction in equipment cost: The traditional laboratory configuration (PCR instrument + electrophoresis system + gel imaging) costs about 80,000 - 150,000 yuan, while the overall cost of this invention is controlled within 20,000 yuan, a reduction of more than 75%; Inexpensive consumables: The cost of a single detection using a microfluidic chip is ≤ 15 yuan (including magnetic beads, enzyme preparations, and fluorescent dyes), saving 60% - 85% compared to commercial kits (50 - 100 yuan per time).

[0022] It realizes "fast, accurate, simple, and cost-saving" in wheat molecular marker detection, completely solving the industry pain points of the traditional method with a long cycle, high cost, and dependence on laboratories, providing convenience for wheat breeding, planting, and market supervision. Brief Description of the Drawings

[0023] Figure 1 It is a schematic structural diagram of the present invention.

[0024] Figure 2 It is a schematic structural diagram of the partial cross-section of the present invention.

[0025] Figure 3 It is a schematic top view structural diagram of the sample placement box of the present invention.

[0026] Figure 4 It is a schematic structural diagram of the sampling power pump assembly of the present invention.

[0027] In the figure: 1. Instrument box body; 2. Moving wheels; 3. Wheat sample crushing chamber; 4. Nucleic acid extraction module; 5. Microfluidic amplification and detection module; 6. Handheld handle; 7. Detection and analysis integrated machine; 8. Result output port; 9. Sample placement box; 91. Longitudinal partition board; 92. Transverse partition board; 93. Mechanical crushing chamber; 94. Ultrasonic crushing chamber; 95. Hand pull handle; 10. Battery; 11. Sampling power pump assembly; 111. Suction pump; 112. Connection conduit; 113. Left conduit; 114. Right conduit; 115. Expander; 116. Detection probe; 12. Ultrasonic generator; 13. Data printer. Detailed Description of the Invention

[0028] The following further describes the present invention with reference to the accompanying drawings: Embodiment

[0029] As shown in the atta Figure 1 ched to the attaFigure 4 As shown in the figure, the present invention provides a portable rapid analyzer for wheat molecular marker detection, which includes an instrument box body 1, moving wheels 2, a wheat sample crushing chamber 3, a nucleic acid extraction module 4, a microfluidic amplification detection module 5, a hand-held handle 6, a detection and analysis integrated machine 7, a result output port 8, a sample placement box 9, a storage battery 10, a sampling power pump assembly 11, an ultrasonic generator 12 and a data printer 13. The moving wheels 2 are respectively bolted to the four corners of the lower part of the instrument box body 1; the moving wheels 2 are specifically silent rubber universal wheels with brake pads; the wheat sample crushing chamber 3 is integrally arranged at the lower part of the instrument box body 1; the nucleic acid extraction module 4 and the microfluidic amplification detection module 5 are screwed to the upper side of the front part of the instrument box body 1 from bottom to top; the microfluidic amplification detection module 5 contains a freeze-dried reagent cartridge pre-loaded with DNA polymerase and dNTPs; the nucleic acid extraction module 4 is internally provided with enzyme-catalyzed reaction reagents; the hand-held handle 6 is screwed to the middle position of the upper rear side of the instrument box body 1; the detection and analysis integrated machine 7 is screwed to the upper front side of the instrument box body 1; the result output port 8 is screwed to the middle position of the upper right side of the instrument box body 1; the sample placement box 9 is inserted into the wheat sample crushing chamber 3 and is located at the lower side of the instrument box body 1; the storage battery 10 is snap-connected to the bottom position inside the instrument box body 1; the sampling power pump assembly 11 is screwed to the inside of the instrument box body 1; the ultrasonic generator 12 is screwed to the left and right sides of the lower part of the instrument box body 1; the data printer 13 is screwed to the upper right side inside the instrument box body 1.

[0030] In the above-mentioned implementation scheme, specifically, the sample placement box 9 includes a longitudinal partition plate 91, a transverse partition plate 92, a mechanical crushing chamber 93, an ultrasonic crushing chamber 94 and a hand-pulling handle 95. The longitudinal partition plate 91 and the transverse partition plate 92 are cross-connected and clamped inside the sample placement box 9; the front side of the sample placement box 9 is divided into a mechanical crushing chamber 93; the rear side of the sample placement box 9 is divided into an ultrasonic crushing chamber 94; the hand-pulling handle 95 is screwed to the middle position of the upper front side of the sample placement box 9.

[0031] In the above-mentioned implementation scheme, specifically, the sampling power pump assembly 11 includes a suction pump 111, a connecting conduit 112, a left conduit 113, a right conduit 114, an expander 115 and a detection probe 116. The connecting conduit 112 is threadedly connected to the inlet end of the suction pump 111; the left conduit 113 and the right conduit 114 are respectively threadedly connected to the left and right sides of the lower part of the connecting conduit 112; the output ends of the expander 115 are adhesively connected to the lower parts of the left conduit 113 and the right conduit 114 respectively; the detection probes 116 are respectively threadedly connected to the lower ends of the left conduit 113 and the right conduit 114.

[0032] In the above embodiments, specifically, the sample placement box 9 is inserted into the interior of the wheat sample crushing chamber 3, and the ultrasonic generators 12 are located on both sides of the ultrasonic crushing chamber 94.

[0033] In the above embodiments, specifically, the data printer 13 and the result output port 8 are located on the same horizontal line and are connected to each other.

[0034] In the above embodiments, specifically, the suction pump 111 and the telescopic device 115 are respectively connected to the inner wall of the instrument box body 1 by screw connections; ventilation holes are provided on the upper and lower sides of the front parts of the left conduit 113 and the right conduit 114.

[0035] In the above embodiments, specifically, the air outlet end of the suction pump 111 is sleeved with a connecting pipeline, and the connecting pipeline is respectively connected to the nucleic acid extraction module 4, the microfluidic amplification detection module 5 and the detection and analysis all-in-one machine 7; the ultrasonic generator 12 integrates ultrasonic crushing (frequency 40 kHz ± 5%).

[0036] In the above embodiments, specifically, the detection and analysis all-in-one machine 7 adopts a replaceable chip design (the chip contains pre-loaded primers, storage temperature 4 - 25 °C), and based on semiconductor temperature control (heating rate 10 °C / s), 40 cycles of amplification can be completed in 15 minutes.

[0037] In the above embodiments, specifically, the nucleic acid extraction module 4 and the microfluidic amplification detection module 5 have a built-in wheat variety database (containing ≥ 100 known variety marker characteristics).

[0038] In the experimental examples, 50 mg of wheat was placed in the sample placement box 9 and the ultrasonic crushing chamber 94, and ultrasonic crushing was started (power 50 W, 30 seconds); analysis was performed through the nucleic acid extraction module 4, the microfluidic amplification detection module 5 and the detection and analysis all-in-one machine 7.

[0039] Experimental data: 1. Comparative test table: | Index | This invention | Laboratory gold standard | Commercially available portable device |---------------|-------------|--------------|--------------| | Detection time | 25 min | 6 h | 1.5 h | | Accuracy rate | 99.2% | 100% | 92% | | Operational complexity | 3 steps | 12 steps | 6 steps | 2. Environmental test report: Continuously run 50 times under the environment of 85% humidity and 35 °C, and the result deviation < 5%.

[0040] The present invention realizes "fast, accurate, simple and cost-saving" for wheat molecular marker detection, thoroughly solves the industry pain points of the traditional method with long cycle, high cost and dependence on laboratories, and provides a revolutionary tool for wheat breeding, planting and market supervision.

[0042] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0043] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A portable and rapid analyzer for wheat molecular marker detection, characterized in that, The portable rapid analyzer for wheat molecular marker detection includes an instrument box body (1), moving wheels (2), a wheat sample crushing chamber (3), a nucleic acid extraction module (4), a microfluidic amplification detection module (5), a hand handle (6), a detection and analysis all-in-one machine (7), a result output port (8), a sample placement box (9), a storage battery (10), a sampling power pump assembly (11), an ultrasonic generator (12) and a data printer (13). The moving wheels (2) are respectively bolted to the four lower corners of the instrument box body (1); the moving wheels (2) are specifically silent rubber universal wheels with brake pads; the wheat sample crushing chamber (3) is integrally arranged at the lower part of the instrument box body (1); the nucleic acid extraction module (4) and the microfluidic amplification detection module (5) are screwed to the upper side of the front part of the instrument box body (1) from bottom to top; the hand handle (6) is screwed to the middle position at the upper rear side of the instrument box body (1).

2. The portable rapid analyzer for wheat molecular marker detection according to claim 1, characterized in that, The detection and analysis all-in-one machine (7) is screwed to the upper front side of the instrument box body (1); the result output port (8) is screwed to the middle position at the upper right side of the instrument box body (1); the sample placement box (9) is inserted into the wheat sample crushing chamber (3) and is located at the lower side of the instrument box body (1); the storage battery (10) is snap-connected to the inner bottom position of the instrument box body (1); the sampling power pump assembly (11) is screwed to the inside of the instrument box body (1); the ultrasonic generator (12) is screwed to the left and right sides at the lower part of the instrument box body (1); the data printer (13) is screwed to the upper right side inside the instrument box body (1).

3. The portable rapid analyzer for wheat molecular marker detection according to claim 1, characterized in that, The sample placement box (9) includes a longitudinal partition board (91), a transverse partition board (92), a mechanical crushing chamber (93), an ultrasonic crushing chamber (94) and a hand pull handle (95). The longitudinal partition board (91) and the transverse partition board (92) are cross-connected and clamped inside the sample placement box (9); the front side of the sample placement box (9) is divided into a mechanical crushing chamber (93); the rear side of the sample placement box (9) is divided into an ultrasonic crushing chamber (94); the hand pull handle (95) is screwed to the middle position at the upper front side of the sample placement box (9).

4. The portable rapid analyzer for wheat molecular marker detection according to claim 1, characterized in that, The sampling power pump assembly (11) includes a suction pump (111), a connecting conduit (112), a left conduit (113), a right conduit (114), a telescopic device (115) and a detection probe (116). The connecting conduit (112) is threadedly connected to the inlet end of the suction pump (111); the left conduit (113) and the right conduit (114) are respectively threadedly connected to the left and right sides at the lower part of the connecting conduit (112); the output ends of the telescopic device (115) are adhesively arranged with the lower parts of the left conduit (113) and the right conduit (114) respectively; the detection probes (116) are respectively threadedly connected to the lower ends of the left conduit (113) and the right conduit (114).

5. The portable rapid analyzer for wheat molecular marker detection according to claim 3, wherein, The sample placement box (9) is inserted inside the wheat sample crushing chamber (3), and the ultrasonic generators (12) are located on both sides of the ultrasonic crushing chamber (94).

6. The portable rapid analyzer for wheat molecular marker detection according to claim 1, characterized in that, The data printer (13) and the result output port (8) are located on the same horizontal line and are connected to each other.

7. The portable rapid analyzer for wheat molecular marker detection according to claim 4, characterized in that, The suction pump (111) and the expander (115) are respectively connected to the inner wall of the instrument box body (1) by screw connections; vent holes are provided on the upper and lower sides of the front parts of the left conduit (113) and the right conduit (114).

8. The portable rapid analyzer for wheat molecular marker detection according to claim 1, characterized in that The microfluidic amplification detection module (5) contains a freeze-dried reagent cartridge pre-loaded with DNA polymerase and dNTPs; the ultrasonic generator (12) integrates ultrasonic crushing (frequency 40 kHz ± 5%).

9. The portable rapid analyzer for wheat molecular marker detection according to claim 1, characterized in that, The nucleic acid extraction module (4) and the microfluidic amplification detection module (5) have a built-in wheat variety database (including ≥ 100 known variety marker characteristics); the nucleic acid extraction module (4) has an enzyme-catalyzed reaction reagent built-in.

10. The portable rapid analyzer for wheat molecular marker detection according to claim 4, wherein The air outlet end of the suction pump (111) is sleeved with a communication pipeline, and the communication pipeline is respectively connected to the nucleic acid extraction module (4), the microfluidic amplification detection module (5) and the detection and analysis all-in-one machine (7).

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