CN115093961A - Multi-volume liquid drop digital LAMP nucleic acid absolute quantitative detection device and method and application - Google Patents
Multi-volume liquid drop digital LAMP nucleic acid absolute quantitative detection device and method and application Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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Abstract
The invention relates to a multi-volume liquid drop digital LAMP nucleic acid absolute quantitative detection device and a method and application thereof, wherein the detection device comprises a detection base, a piezoelectric sensor fixing component, a temperature control heating platform and a smart phone optical detection component are sequentially arranged on the surface of the detection base from one side to the other side, a light-shading cover is further arranged on the detection base, and the piezoelectric sensor fixing component, the temperature control heating platform and the smart phone optical detection component are covered in the light-shading cover; the light shading cover is provided with a light through hole; the light through hole is correspondingly arranged on the optical detection component of the smart phone. The device is low in manufacturing cost and convenient to operate, the detection method based on the device has the advantages of high sensitivity, independence on Ct value, high tolerance, short time consumption, capability of regulating and controlling generation of liquid drops with different sizes, capability of carrying out absolute quantitative detection on nucleic acid in a wide linear range and the like, and has a good application prospect in the aspect of on-site instant detection of pathogenic microorganisms.
Description
Technical Field
The invention relates to the technical field of biological and medical detection, in particular to a device and a method for absolutely and quantitatively detecting LAMP nucleic acid with multiple liquid drops and application.
Background
The nucleic acid is a carrier of genetic information in a living body (CHEN, K, et al.,2016), realizes rapid, accurate and high-sensitivity quantitative analysis of the nucleic acid, and has very important significance for disease diagnosis and treatment, public health and food safety, basic biological research and the like. The conventional nucleic acid detection method amplifies a specific DNA fragment (HUANG, H S, et al, 2018) based on Polymerase Chain Reaction (PCR), analyzes the amplified product by agarose gel electrophoresis or monitors the amplification process by real-time fluorescence change, and has the advantages of high sensitivity, good specificity and the like. However, since PCR comprises three steps of denaturation, annealing and extension which are performed at different temperatures, 1.5-2h is usually required for amplification of target nucleic acid, and the reaction time is long; amplification requires a precise temperature control system, electrophoresis or fluorescence monitoring relies on additional analytical instruments, and needs to be performed in standard laboratories. In order to realize simple and rapid nucleic acid detection, some nucleic acid analysis methods based on isothermal nucleic acid amplification technology have been developed in recent years. Wherein, the Loop-mediated isothermal amplification (LAMP) technology designs four (or six) primers aiming at six regions of a target gene, uses a strand displacement DNA polymerase to carry out amplification reaction, can realize high-efficiency amplification (NONONOMI, T, et al.,2000) of the target nucleic acid within 30-60min under the isothermal (60-65 ℃) condition, has quick reaction, simple operation and good specificity, and is widely applied to the instant detection of pathogen nucleic acid. At present, the LAMP-based nucleic acid analysis method is mainly used for detection based on chromogenic or fluorescent reaction in a test tube (KIM, J K, et al.,2021), a test strip (POIRIER, A C, et al.,2022) or a microfluidic chip (ZHOU, L, et al.,2020), and the systems have lower requirements on instruments and operation and are more suitable for practical application and popularization; however, these systems often have the problems of relatively high detection limit, insufficient sensitivity, etc., and usually only qualitative or semi-quantitative results can be obtained, and accurate quantitative analysis of the nucleic acid content in the sample cannot be realized.
Digital nucleic acid amplification detection (dNAD) is an absolute nucleic acid molecule quantification technology, a sample system is randomly and uniformly dispersed into thousands of independent reaction units, each reaction unit contains at most one nucleic acid target molecule to be detected as far as possible, after the nucleic acid target molecules are amplified in the reaction units, the reaction unit with a fluorescence signal is counted as 1, the reaction unit without the fluorescence signal is counted as 0 by detecting the fluorescence signal value of each reaction unit, and the absolute copy number (HINDSON, B J, et al, 2011) of the nucleic acid target molecules in the sample system can be calculated according to the number of the reaction units with or without the fluorescence signals and the Poisson distribution principle. The technology can carry out absolute quantification without depending on a standard curve, has higher sensitivity and accuracy compared with a real-time fluorescence quantification method, and can effectively avoid the influence of a reaction inhibitor. Depending on the implementation of its individual units, dnads can be divided into two classes (YUAN, H, et al, 2019) of micro-Droplet based micro-fluidics and micro-array based on chip micro-fluidics. At present, the digital PCR technology based on the principle is widely applied to the fields of pathogenic microorganism detection, food safety monitoring, genome analysis, clinical examination and the like
J,et al.,2016,TAO,Y,et al.,2020,WANG,J,et al.,2021,ZHANG,M,et al.,2021)。
The digital nucleic acid amplification detection is combined with LAMP technology, so that the reaction time can be shortened, the cyclic heating is avoided, and the high-sensitivity absolute quantitative analysis of the target nucleic acid is realized. However, the currently established digital LAMP method still faces the following disadvantages: firstly, the existing micro-droplet or micro-array method mostly depends on a micro-fluidic chip to carry out sample system distribution, the manufacturing process of the micro-fluidic chip is complex, the cost is higher, and the operation often needs matching equipment such as a precision pressure pump and the like, so that the practical application and popularization of the micro-fluidic chip are limited; secondly, the fluorescence signal detection of the reaction unit depends on expensive and precise optical instruments, such as a fluorescence microscope, laser-induced fluorescence detection and the like, so that the detection cost is greatly improved; thirdly, the existing digital LAMP method usually uses a single fixed reaction unit volume, the detection linear range is limited, and the linear range can be expanded only by increasing the number of reaction units, which puts higher requirements on a unit distribution and detection system; finally, in the existing method, the samples, the enzymes, the primers and the like are premixed and then distributed to the reaction units, and if the premixing time is too long, the background signal is increased, the differentiation and counting of the positive and negative units are interfered, and the accuracy of the detection result is influenced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a multi-volume droplet digital LAMP nucleic acid absolute quantitative detection device, a method and application, the method utilizes the generation of micro-droplets driven by sound waves, and the device is simple, the operation is convenient and fast, and the complex micro-fluidic chip manufacturing process is not relied on; the fluorescent signal is obtained by photographing through the smart phone and then is processed and analyzed, so that the detection cost is low; the linear range of detection can be greatly improved through the multi-volume liquid drops under the condition of not increasing the number of reaction units, and the method has strong operability, short time consumption and good application prospect in the field instant detection of pathogenic microorganisms.
The scheme for solving the technical problems is as follows: the device for absolutely and quantitatively detecting the LAMP nucleic acid of the multiple liquid drops comprises a detection base, wherein a piezoelectric sensor fixing component, a temperature control heating platform and a smartphone optical detection component are sequentially arranged on the surface of the detection base from one side to the other side, a light-shading cover is further arranged on the detection base, and the piezoelectric sensor fixing component, the temperature control heating platform and the smartphone optical detection component are covered in the light-shading cover; the light shading cover is provided with a light through hole; the light through hole is correspondingly arranged on the optical detection component of the smart phone;
the piezoelectric sensor fixing assembly comprises an inverted L-shaped support frame, a vertical support frame and a horizontal support frame; the horizontal supporting frame is positioned above the temperature control heating platform;
a rotary piezoelectric sensor fixing platform is arranged on the horizontal support frame, and grooves for mounting a vibration tip device are arranged on two side walls of the rotary piezoelectric sensor fixing platform at intervals;
the vibration tip device comprises a micro pipeline with a stretched tip, a sample inlet at the top of the micro pipeline is communicated with a main pipe of a Y-shaped three-way pipe, and two branch pipes of the Y-shaped three-way pipe are respectively externally connected with a first sample conveying hose and a second sample conveying hose; the Y-shaped three-way pipe is provided with a piezoelectric sensor which is electrically connected with the portable signal generator; the micro-pipeline can adopt a glass capillary tube with a stretched tip, the outer diameter of the glass capillary tube is 1mm, the inner diameter of the glass capillary tube is 0.58mm, the stretched tip is a conical opening, and the outer diameter of the glass capillary tube is 15-60 mu m; the portable signal generator is electrically connected to generate sine waves, square waves and trigonometric functions and can generate analog and digital modulation signals;
the optical detection assembly of the smart phone comprises a mounting base, wherein an object stage and an optical detection support frame are arranged on the mounting base, an LED lamp light source is arranged on the end wall of the object stage, an exciting light filter and a light guide plate are arranged on the emitting route of the LED lamp light source, the LED lamp light source is arranged in a socket on the side wall of the object stage, and the light guide plate is arranged in the object stage; the optical detection support frame is provided with an optical detection seat, and a lens and an emission light filter are arranged on the optical detection seat;
the optical detection seat is arranged above the objective table.
Preferably, the temperature control heating platform comprises a PTC electric heating plate, a temperature measuring hole and two power interfaces are arranged on the side wall of the PTC electric heating plate, and the temperature measuring hole is electrically connected with the digital display temperature controller.
Preferably, the PTC heating plate is fixed to the sensing base by heat insulation fixing plates at both sides.
Preferably, the vertical support frame is an upper telescopic frame and a lower telescopic frame, a mounting seat which moves up and down is arranged on the vertical support frame, the horizontal support frame is inserted into the mounting seat, a support rod nesting hole is formed in the rotary piezoelectric sensor fixing platform, and the rotary piezoelectric sensor fixing platform is nested on the horizontal support frame through the support rod nesting hole; the rotary piezoelectric sensor fixing platform is characterized in that mounting columns are arranged on two sides of the top surface of the rotary piezoelectric sensor fixing platform at intervals, and raised sample conveying hose placing holes are formed in the mounting columns.
Preferably, a plurality of threaded holes are arranged on the detection base.
The method for absolutely and quantitatively detecting the multiple-volume liquid drop digital LAMP nucleic acid by using the device comprises the following steps:
1) sample processing
Extracting and purifying nucleic acid after sample pretreatment, and preparing the nucleic acid, dye, specific primer, partial LAMP buffer solution and ribozyme-free water into liquid a; mixing enzyme and partial LAMP buffer solution to prepare liquid b;
2) production of micro-droplets
The liquid a and the liquid b enter the Y-shaped three-way pipe through the first sample conveying hose and the second sample conveying hose;
placing a container filled with an oil phase on the PTC electric heating plate, and adjusting the position of the stretched tip of the micro-pipeline to immerse the stretched tip into the liquid surface of the oil phase;
starting a portable signal generator (regulating signal frequency, output voltage and pulse frequency, converting an input electric signal into an acoustic signal by a piezoelectric sensor and outputting the acoustic signal to cause the stretching tip of the micro-pipeline to vibrate, and forming a negative pressure region at the tip by induced vortex), and enabling the liquid a and the liquid b to enter a Y-shaped three-way pipe through a first sample conveying hose and a second sample conveying hose;
then mixing the mixture at the tip and injecting the mixture into the oil phase to form monodisperse LAMP reaction micro-droplets;
3) micro-droplet isothermal amplification
Removing the vibration tip device, starting the temperature control heating platform, and maintaining the constant temperature of 60-65 ℃ for amplification for 30-60 min;
4) photographing device
Transferring the container with the collected micro liquid drops to an objective table in the optical detection assembly of the smart phone, placing the smart phone above the device, enabling a camera of the smart phone to be opposite to the light through hole, starting an LED light source, starting a shooting function of the smart phone, rotating the thread rotating sleeve to adjust an imaging focal length, collecting bright field images of the liquid drops under the condition that the optical filters are not inserted, then inserting the exciting light optical filters and the emitting light optical filters, and collecting fluorescence images of the liquid drops.
5) Analyzing and calculating the properties and the quantity of the micro-droplets
Using Image J software to carry out Image coding, filtering, edge detection and other processing on the Image, identifying and reading the average fluorescence intensity value of the negative liquid drop under the fluorescence as a threshold value F,
if the average fluorescence intensity value of the liquid drop is greater than F, the liquid drop is positive;
if the average fluorescence intensity value of the liquid drop is less than or equal to F, the liquid drop is a negative liquid drop;
counting the number of liquid drops under a bright field, the number of positive liquid drops under fluorescence, measuring the average diameter of the liquid drops, calculating the detection rate and carrying out binarization processing, and calculating the concentration or copy number of a sample to be detected by using the following formula according to the total number of the liquid drops, the number of the positive liquid drops and the average diameter of the liquid drops by utilizing a Poisson distribution principle according to the counted total number of the liquid drops, the number of the positive liquid drops and the average diameter of the liquid drops:
wherein d is the average diameter of the droplets in μm, and V is the average volume of the droplets in μm;
c is the concentration or copy number of the nucleic acid to be detected, and the unit is copies/mu L;
m is the dilution factor of the sample;
p is the number of positive droplets and the unit is one;
r is the total number of droplets and is expressed in units of units.
Preferably, in the step 2), the sample introduction volume ratio of the liquid a to the liquid b is 1:1, the liquid a is a mixed solution of the nucleic acid sample to be detected, the dye, the specific primer, part of the LAMP buffer solution and the ribozyme-free water, and the liquid b is a mixed solution of the DNA polymerase with the strand displacement activity and part of the LAMP buffer solution.
Preferably, in the step 2), the oil phase is prepared from a mixture of 100: 5-7 of mineral oil in molecular biology and surfactant.
Preferably, in the step 2), the signal frequency of the portable signal generator is adjusted within a range of 85-110kHz, the output voltage is adjusted within a range of 3-20Vpp, the pulse frequency is adjusted within a range of 1-5000Hz, and the diameter of the generated micro-droplets is within a range of 10-600 μm.
Preferably, in step 3), after the micro-droplets are produced, the micro-droplets need to be cleaned, and the cleaning steps are as follows:
respectively and sequentially injecting a nucleic acid pollution clearing agent and high-pressure sterilized water into the first sample conveying hose and the second sample conveying hose, replacing the oil phase with the high-pressure sterilized water, still immersing the stretched tip of the micro-pipeline in the high-pressure sterilized water, starting the portable signal generator, and cleaning the Y-shaped three-way pipe and the micro-pipeline.
Preferably, a micro-droplet collecting container is used as a container for collecting micro-droplets and containing an oil phase, wherein the micro-droplet collecting container is formed by bonding Polydimethylsiloxane (PDMS) and a glass sheet subjected to hydrophobic treatment;
the PDMS is prepared by uniformly mixing a prepolymer, an initiator and mineral oil in a ratio of 10:1:1, degassing, curing in a 65 ℃ oven for 2 hours, and cutting or punching;
the hydrophobization treatment steps of the slide slice are as follows: after washing with 75% alcohol for 30s, pre-treating with 0.1mol/L sodium hydroxide solution for 20min and air-drying, then reacting in 0.1% octadecyl trimethylsilane in toluene for 2h and air-drying, finally drying at 65 ℃ for 2h, and washing with absolute ethanol for 30 s.
The invention also provides an application of the detection device in detecting the porcine epidemic diarrhea virus.
The invention has the following beneficial effects:
(1) according to the invention, the liquid a and the liquid b are conveyed into the micro-pipeline by using the Y-shaped three-way pipe, and the LAMP reaction mixed liquid to be detected (the mixed liquid of the liquid a and the liquid b) is mixed and driven by using sound waves to generate liquid drops, so that the influence of room temperature on the LAMP reaction mixed liquid is avoided, and the LAMP reaction amplification background is reduced;
(2) the method and the device for generating the acoustic wave-driven micro-droplets can generate multi-volume monodisperse droplets at high flux, can regulate and control the generation of the droplets in a wide scale range (10-600 mu m) in real time, accurately and flexibly without complex chip structure design and operation steps;
(3) the main part for generating the acoustic wave-driven micro liquid drops only comprises the tip stretching micro pipeline controlled by the piezoelectric sensor, the Y-shaped three-way pipe and the portable signal generator, and has the advantages of simple system, convenience and quickness in operation, low cost and no need of complex, expensive and huge liquid drop generation supporting equipment;
(4) compared with conventional PCR, qPCR and digital PCR, the digital LAMP technology for detecting the multiple-volume droplets has the advantages of high sensitivity, no dependence on Ct value, high tolerance, reaction time of only 30-60min, capability of absolutely and quantitatively detecting nucleic acid in a wide linear range and the like;
(5) under the condition of needing to detect for many times, the invention can detect the micro reaction mixed liquid (25-100 mu L) to be detected again only by washing the Y-shaped three-way pipe, the tip-stretched micro pipeline and the oil phase (only 1-3ml are needed for four chambers) with a small amount of high-pressure sterilized water, thereby reducing the reagent cost;
(6) the device integrates the generation, amplification and fluorescence signal detection of the liquid drops into a set of multi-volume liquid drop digital LAMP detection device, has the advantages of low manufacturing cost, convenient operation (the generation of liquid drops with different sizes can be regulated and controlled by regulating and controlling the portable signal generator), short time consumption and good application prospect in the aspect of on-site instant detection of pathogenic microorganisms.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to make the technical solutions of the present invention practical in accordance with the contents of the specification, the following detailed description is given of preferred embodiments of the present invention with reference to the accompanying drawings. The detailed description of the present invention is given in detail by the following examples and the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention and do not constitute a limitation of the invention. In the drawings:
FIG. 1 is a schematic structural diagram of a digital LAMP detection device for multiple liquid drops based on acoustic drive according to the present invention;
FIG. 2 is a front view of FIG. 1;
FIG. 3 is a schematic structural view of a detection base and a piezoelectric sensor fixing device according to the present invention;
FIG. 4 is a front view of a piezoelectric sensor mounting assembly of the present invention;
FIG. 5 is a left side view of the piezoelectric sensor mounting assembly of the present invention;
FIG. 6 is a schematic view of the vibrating tip of the present invention;
FIG. 7 is a schematic view of the structural principle of the optical detection assembly of the smart phone of the present invention
FIG. 8 is a right side view of FIG. 7;
FIG. 9 is a front view of FIG. 7;
FIG. 10 is a schematic view of a temperature controlled heating stage according to the present invention;
FIG. 11 is a top view of FIG. 10;
FIG. 12 is a schematic view showing the connection between the digital display temperature controller and the PTC electric heating plate according to the present invention;
FIG. 13 is a schematic view of a micro-droplet collection container according to the present invention;
FIG. 14 is a top view of FIG. 13;
FIG. 15 is a flow chart of the digital LAMP detection method for multiple liquid drops based on acoustic wave driving according to the present invention;
fig. 16 is an optical microscope image of four droplets with different diameters formed by the acoustic wave drive-based multi-volume droplet digital LAMP device of the present invention in example 2;
fig. 17 is a fluorescence diagram of a part of droplets formed by detecting a porcine epidemic diarrhea virus CV777 strain by using a multi-volume droplet digital LAMP method and device based on acoustic drive in example 2 of the invention;
fig. 18 is a fluorescence image of a part of droplets formed by the digital LAMP method and device for multiple bulk droplets based on acoustic drive in the detection of a piglet fecal sample suspected of suffering from porcine epidemic diarrhea in example 3 of the present invention.
In the drawings, the reference numbers indicate the following list of parts:
1. vibrating the tip device; 2. a temperature controlled heating platform; 3. the smart phone optical detection component; 4. detecting a base; 11. an aqueous phase; 12. an oil phase; 13. a micro-droplet collection vessel; 14. a portable signal generator; 101. a Y-shaped three-way pipe; 102. a micro-pipe; 103. a piezoelectric sensor; 104. a first sample transfer hose; 105. a second sample delivery hose; 106. a single four-chamber reservoir; 107. a multi-chamber liquid storage tank; 108. PDMS; 109. a glass sheet; 21. a digital display temperature controller; 22. a PTC electrical heating plate; 201. a heat insulation fixing plate; 202. a temperature measuring hole; 203. a power interface; 31. a smart phone; 32. an LED light source; 33. an excitation light filter; 34. an emission light filter; 35. a light guide plate; 36. a lens; 37. an optical detection support frame; 301. an object stage; 302. adjusting the threaded knob up and down; 303. a left and right adjusting threaded knob; 41. a piezoelectric sensor fixing component; 42. a light-resistant cover; 401. a vertical support frame; 402. a horizontal support frame; 403. rotating the piezoelectric susceptor fixed platform; 404. a light through hole; 405. a mounting seat; 406. a sample conveying hose placing hole; 407. the supporting rod is embedded in the hole; 408. and (6) a groove.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
Example 1
As shown in fig. 1-2, the invention provides a multiple-volume droplet digital LAMP nucleic acid absolute quantitative detection device, which comprises a detection base 4 with a plurality of threaded holes arranged on the surface, wherein a piezoelectric sensor fixing component 41, a temperature control heating platform 2 and a smart phone optical detection component 3 are sequentially arranged on the surface of the detection base 4 from one side to the other side, a light-shielding cover 42 is further arranged on the detection base 4, and the piezoelectric sensor fixing component 41, the temperature control heating platform 2 and the smart phone optical detection component 3 are covered in the light-shielding cover 42; the light-shading cover 42 is provided with a light-through hole 404; the light through hole 404 is correspondingly arranged on the optical detection component 3 of the smart phone, and the smart phone 31 takes a picture through the light through hole 404.
As shown in fig. 3-5, the piezoelectric sensor fixing assembly 41 includes an inverted L-shaped support frame including a vertical support frame 401 and a horizontal support frame 402; and the horizontal support frame 402 is positioned above the temperature control heating platform 2; a rotary piezoelectric receptor fixing platform 403 is arranged on the horizontal support frame 402, and grooves 408 for mounting the vibration tip device 1 are arranged on two side walls of the rotary piezoelectric receptor fixing platform 403 at intervals; the vertical support frame 401 is an up-down telescopic frame, an installation seat 405 which moves up and down is arranged on the vertical support frame 401, the horizontal support frame 402 is inserted into the installation seat 405, a support rod embedding hole 407 is formed in the rotary piezoelectric receptor fixing platform 403, and the rotary piezoelectric receptor fixing platform 403 is embedded in the horizontal support frame 402 through the support rod embedding hole 407; the two sides of the top surface of the rotary piezoelectric sensor fixing platform 403 are provided with mounting columns at intervals, and the mounting columns are provided with convex sample conveying hose placing holes 406.
As shown in fig. 6, the vibrating tip 1 includes a micro-pipe 102 with a stretched tip, the stretched tip of the micro-pipe is immersed in the oil phase 12, a feed inlet at the top of the micro-pipe 102 is communicated with a main pipe of a Y-shaped three-way pipe 101, and two branch pipes of the Y-shaped three-way pipe 101 are respectively connected with a first sample-transporting hose 104 and a second sample-transporting hose 105 filled with a water phase 11 (liquid a and liquid b); a piezoelectric sensor 103 is arranged on the Y-shaped three-way pipe 101, and the piezoelectric sensor 103 is electrically connected with the portable signal generator 14; the microchannel 102 may be a glass capillary with a stretched tip, an outer diameter of 1mm and an inner diameter of 0.58 mm; the stretching tip of the micro-pipe 102 is centrally, vertically and downwards adhered to a metal plate of the piezoelectric sensor 103, the tip of the stretching tip protrudes 3cm from the lower edge of the piezoelectric sensor 103, the tip of the stretching tip is a conical opening, and the outer diameter of the stretching tip is 15-60 mu m; the electrically connected portable signal generator 14 can generate sine waves, square waves and trigonometric functions, can generate analog and digital modulation signals, and can also adjust electrical signals such as signal frequency, output voltage, pulse frequency and the like, so that the piezoelectric sensor 103 generates different sound wave signals to drive the micro-pipe 102 to generate liquid drops with different sizes.
As shown in fig. 7-9, the optical detection assembly 3 of the smartphone includes a mounting base on which an object stage 301 and an optical detection support frame 37 are disposed. An LED lamp light source is arranged on the end wall of the object stage 301, an exciting light filter 33 and a light guide plate 35 are arranged on the emitting route of the LED lamp light source, the exciting light filter 33 is arranged in a socket on the side wall of the object stage 301, and the light guide plate 35 is arranged in the object stage 301; the optical detection support frame 37 is provided with an optical detection seat, the optical detection seat is provided with a lens 36 and an emission light filter 34, the optical detection seat is arranged above the objective table 301, and the optical detection support frame 37 is further provided with an up-down adjusting threaded knob 302 and a left-right adjusting threaded knob 303 for adjusting the focal length. The stage 301 carries a micro-droplet collection container 13 for the amplified droplets.
The LED light source 32 screens out light with wavelength capable of exciting the calcein dye through the exciting light filter 33 and the light guide plate 35, uniformly irradiates liquid drops in the micro-liquid drop collecting container 13, fluorescence excited in the liquid drops is received and photographed by the smart phone 31 through the emitting light filter 34, the lens 36 and the light through hole 404, Image J software is used for collecting images to carry out correction, edge detection, counting of the number of the liquid drops in a bright field, identification and counting of the number of the positive liquid drops in fluorescence, detection rate calculation and binarization processing, measurement of the diameter of the liquid drops, data processing analysis and calculation, and display of the concentration or copy number of a sample to be measured.
As shown in fig. 10-12, the temperature control heating platform 2 includes a PTC electric heating plate 22, a temperature measuring hole 202 and two power interfaces 203 are arranged on the side wall of the PTC electric heating plate 22, and the temperature measuring hole 202 is electrically connected with the digital display temperature controller 21; the PTC electric heating plate 22 is fixed on the detection base 4 through the heat insulation fixing plates 201 on both sides, and the micro-droplet collecting container 13 collecting droplets is placed on the PTC electric heating plate 22 and heated to amplify.
As shown in fig. 13-14, the micro-droplet collecting container 13 can be used to contain the oil phase 12 and collect the droplets outputted by the micro-pipe 102 under vibration, and the micro-droplet collecting container 13 is formed by bonding PDMS108 and the hydrophobized glass sheet 109; either a single four-chamber reservoir 106 or a 4 x 4 array of multi-chamber reservoirs 107 may be used.
PDMS108 is prepared by uniformly mixing PDMS prepolymer, initiator and mineral oil in a ratio of 10:1:1, degassing, curing in a drying plate at 65 ℃ for 2h, and cutting or punching; the hydrophobization treatment steps of the glass flake 109 are: after the glass sheet 109 is washed by 75% alcohol for 30s, the glass sheet 109 is pretreated by 0.1mol/L sodium hydroxide solution for 20min, then the glass sheet 109 is placed in 0.1% octadecyl trimethyl silane toluene solution for reaction for 2h, finally the glass sheet is dried at 65 ℃ for 2h, and the treated glass sheet 109 is washed by 30s in absolute ethyl alcohol, so that the method can be used for preparing the micro-droplet collection container 13.
Example 2
With the device, as shown in fig. 8, the specific steps for absolute quantitative detection of porcine epidemic diarrhea virus CV777 are as follows:
1. extracting RNA of the supernatant of the porcine epidemic diarrhea virus CV777 by using a Trizol method, carrying out reverse transcription on the RNA into DNA, and designing 6 pairs of LAMP specific primers of the porcine epidemic diarrhea virus for detection so as to shorten the amplification time, wherein the sequences of the primers are shown in a table 1:
TABLE 1
2. The DNA containing 4. mu.L of template DNA (estimated template DNA concentration of 10 using TGem Plus full-wavelength spectrophotometer) was prepared in a clean bench 5 copies/. mu.L), 2. mu.L of dye reagent, 5. mu.L of ribozyme-free water, 14. mu.L of LAMP buffer solution as liquid a, and 2. mu.L of Bst polymerase with strand displacement activity, 12. mu.L of primer solution DNA (containing 40pmol FIP, 40pmol BIP, 5pmol F3, 5pmol B3) and 11. mu.L of LAMP reaction mixture as liquid B (the total volume of liquid a and liquid B is 50. mu.L, porcine epidemic diarrhea is induced byLAMP reaction mixed liquor of DNA obtained by reverse transcription of a virus CV777 strain) and a negative control group is set by using sterile and enzyme-free water;
3. fixing two vibration tips 1 in different grooves 408 of the piezoelectric sensor fixing member 41;
4. injecting the liquid a and the liquid b of the negative control group in the step 2 into the Y-shaped three-way pipe 101 through the first sample conveying hose 104 and the second sample conveying hose 105 respectively by using the first vibration tip 1; injecting the liquid a and the liquid b of the positive test group in the step 2 into a Y-shaped three-way pipe 101 through a first sample conveying hose 104 and a second sample conveying hose 105 respectively by using a second vibration tip 1, and injecting an oil phase 12 into a multi-cavity liquid storage tank 107;
5. adjusting the piezoelectric sensor fixing component 41 to enable the tips of the two micro-pipes 102 to be respectively immersed below the liquid levels of the oil phases 12 in different chambers in the multi-chamber liquid storage tank 107;
6. the portable signal generator 14 is connected with the piezoelectric sensor 103 through a lead, and the voltage, the signal frequency and the pulse frequency of the portable signal generator 14 are adjusted, so that the two micro-tubes 102 respectively generate stable monodisperse liquid drops with uniform size of about 490 microns, 240 microns, 113 microns and 53 microns in diameter in the oil phase 12 of different chambers of the multi-chamber liquid storage tank 107, as shown in fig. 16;
7. adjusting the height of the piezoelectric sensor mounting assembly 41 to allow the tips of the two microchannels 102 to exit the multi-chamber reservoir 107, respectively;
8. standing the multi-cavity liquid storage tank 107 for 2min to enable the liquid drops to be flatly paved on the bottom surface of the multi-cavity liquid storage tank 107 as single layer as possible;
9. transferring the multi-cavity liquid storage tank 107 to the PTC electric heating plate 22, adjusting the digital display temperature controller 21 to 65 ℃, and carrying out constant temperature amplification on the liquid drops to be detected in the multi-cavity liquid storage tank 107 for 35min at 65 ℃;
10. transferring the multi-cavity liquid storage tank 107 obtained in the step 9 into the optical detection assembly 3 of the smart phone, turning on the LED light source 32 and adjusting the focal length, facing the camera hole of the smart phone 31 to the light through hole 404, and using the smart phone 31 to respectively shoot images of all liquid drops in the multi-cavity liquid storage tank 107 in a bright field and a fluorescence state, wherein partial images are shown in fig. 17, and after gray processing, positive liquid drops are light white or white and are distinguished from negative liquid drops by relatively obvious fluorescence;
11. performing luminosity correction, coding and edge detection on the droplet image obtained by the step 10, identifying and reading the average fluorescence intensity value of the negative droplets under fluorescence as a threshold value F, identifying the droplets with fluorescence intensity higher than F as positive droplets, counting the number of the droplets under bright field, the number of the positive droplets under fluorescence, measuring the average diameter of the droplets, calculating the detection rate and performing binarization processing, and calculating the concentration or copy number of DNA (deoxyribonucleic acid) obtained by reverse transcription of the porcine epidemic diarrhea virus CV777 strain by using the following formula according to the counted total number of the droplets, the number of the positive droplets and the average diameter of the droplets by utilizing a Poisson distribution principle:
12. the concentration of the DNA obtained by reverse transcription of the porcine epidemic diarrhea virus CV777 strain is 7.7 multiplied by 10 4 copies/μL；
13. After the detection is finished, the liquid drop of the sample to be detected and the oil phase 12 are subjected to harmless treatment and then poured into a corresponding waste liquid box.
Example 3
The method comprises the following specific steps of taking a piglet excrement sample suspected of suffering from porcine epidemic diarrhea, applying the device, and detecting the piglet excrement sample as shown in figure 15:
1. diluting a piglet excrement sample suspected of suffering from porcine epidemic diarrhea by 5-10 times with PBS buffer solution or normal saline, uniformly mixing by using a vortex apparatus, centrifuging for 5min at 12000r/min, taking 200 mu L of supernatant, extracting RNA by using a Trizol method, carrying out reverse transcription to obtain DNA, and designing 4 pairs of LAMP specific primers of porcine epidemic diarrhea virus for detection, wherein the sequences of the primers are shown in Table 2:
TABLE 2
2. Preparing a liquid a containing 4 mu L of template DNA, 2 mu L of dye reagent, 5 mu L of ribozyme-free water and 14 mu L of LAMP buffer solution in an ultra-clean bench, preparing a liquid B containing 2 mu L of Bst polymerase with strand displacement activity, 12 mu L of primer solution DNA (containing 40pmol FIP, 40pmol BIP, 5pmol F3 and 5pmol B3) and 11 mu L of LAMP reaction mixed solution (the LAMP reaction mixed solution of the DNA obtained by reverse transcription of the porcine epidemic diarrhea virus CV777 strain with the total volume of 50 mu L of liquid a and liquid B), and setting a negative control group by using sterile enzyme-free water;
3. fixing the vibration tip 1 in the groove 408 of the piezoelectric sensor fixing member 41;
4. respectively injecting the liquid a and the liquid b of the negative control group in the step 2 into a Y-shaped three-way pipe 101 through a first sample delivery hose 104 and a second sample delivery hose 105, and injecting an oil phase 12 into a single four-cavity liquid storage tank 106;
5. adjusting the piezoelectric sensor mounting assembly 41 so that the tip of the micro-pipe 102 is immersed below the level of the oil phase 12 in the first chamber of the single four-chamber reservoir 106;
6. connecting the portable signal generator 14 with the piezoelectric sensor 103 through a lead, and adjusting the voltage, signal frequency and pulse frequency of the portable signal generator 14, so that the micro-pipe 102 generates stable monodisperse liquid drops with uniform size in the oil phase 12 of the first chamber of the single four-chamber liquid storage tank 106;
7. adjusting the height of the piezoelectric sensor mounting assembly 41 so that the tip of the microchannel 102 exits the single four-chamber reservoir 106;
8. standing the single four-cavity liquid storage tank 106 for 2min to enable the liquid drops to be flatly paved on the bottom surface of the single four-cavity liquid storage tank 106 in a single layer as much as possible;
9. respectively and sequentially injecting a nucleic acid contamination clearing agent and high-pressure sterilizing water into the first sample delivery hose 104 and the second sample delivery hose 105, replacing the oil phase 12 in the second chamber of the single four-chamber liquid storage tank 106 with the high-pressure sterilizing water, immersing the stretching tip of the micro-pipeline 102 in the high-pressure sterilizing water in the single four-chamber liquid storage tank 106, starting the portable signal generator 14, and cleaning the Y-shaped three-way pipe 101 and the micro-pipeline 102;
10. respectively injecting the liquid a and the liquid b of the positive test group in the step 2 into the Y-shaped three-way pipe 101 through a first sample conveying hose 104 and a second sample conveying hose 105;
11. repeating steps 5, 6, 7 and 8 with the third chamber of the single four-chamber reservoir 106 as the droplet collection container of the positive test group;
12. transferring the single four-cavity liquid storage tank 106 to the PTC electric heating plate 22, adjusting the digital display temperature controller 21 to 65 ℃, and amplifying the liquid drops to be detected in the single four-cavity liquid storage tank 106 at the constant temperature of 65 ℃ for 1 h;
13. transferring the single four-cavity liquid storage tank 106 in the step 12 into the optical detection assembly 3 of the smart phone, turning on the LED light source 32 and adjusting the focal length, facing the camera hole of the smart phone 31 to the light through hole 404, using the smart phone 31 to respectively shoot all liquid drop images in the bright field and fluorescence states in the first cavity and the second cavity of the single four-cavity liquid storage tank 106, wherein a partial image is as shown in fig. 18, and after gray level processing, a positive liquid drop is light white or white and is distinguished from a negative liquid drop by more obvious fluorescence;
14. performing luminosity correction, coding and edge detection on the image obtained by the step 10), identifying and reading the average fluorescence intensity value of the negative droplets under fluorescence as a threshold value F, identifying the droplets under fluorescence with the fluorescence intensity larger than F as positive droplets, counting the number of the droplets under bright field and the number of the positive droplets under fluorescence, wherein the negative control group has no nucleic acid amplification and has no positive droplets; the positive test group has nucleic acid amplification and relatively obvious luminous positive liquid drops, which proves that the piglet feces sample contains the porcine epidemic diarrhea virus and the piglet has the porcine epidemic diarrhea. In addition, the image obtained by shooting can be processed as in the step 11) in the embodiment 2, so that the porcine epidemic diarrhea virus in the excrement of the piglets can be quantified, wild virus can be distinguished, and the disease standard can be determined; the method can also be used for the evaluation of the quality content of the vaccine, the clinical evaluation of the animal protection product and the like.
15. After the detection is finished, the liquid drop of the sample to be detected and the oil phase 12 are subjected to harmless treatment and then poured into a corresponding waste liquid box.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner; the present invention may be readily implemented by those of ordinary skill in the art as illustrated in the accompanying drawings and described above; however, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the scope of the invention as defined by the appended claims; meanwhile, any changes, modifications, and evolutions of the equivalent changes of the above embodiments according to the actual techniques of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (10)
1. The LAMP nucleic acid absolute quantitative detection device for the multiple liquid drops is characterized by comprising a detection base (4), wherein a piezoelectric sensor fixing component (41), a temperature control heating platform (2) and a smart phone optical detection component (3) are sequentially arranged on the surface of the detection base (4) from one side to the other side, a light-shading cover (42) is further arranged on the detection base (4), and the piezoelectric sensor fixing component (41), the temperature control heating platform (2) and the smart phone optical detection component (3) are covered in the light-shading cover (42); the light shading cover (42) is provided with a light through hole (404); the light through hole (404) is correspondingly arranged on the optical detection component (3) of the smart phone;
the piezoelectric sensor fixing component (41) comprises an inverted L-shaped support frame which comprises a vertical support frame (401) and a horizontal support frame (402); the horizontal support frame (402) is positioned above the temperature control heating platform (2);
a rotary piezoelectric receptor fixing platform (403) is arranged on the horizontal support frame (402), and grooves (408) for mounting the vibration tip device (1) are arranged on two side walls of the rotary piezoelectric receptor fixing platform (403) at intervals;
the vibration tip device (1) comprises a micro pipeline (102) with a stretched tip, a sample inlet at the top of the micro pipeline (102) is communicated with a main pipe of a Y-shaped three-way pipe (101), and two branch pipes of the Y-shaped three-way pipe (101) are respectively externally connected with a first sample conveying hose (104) and a second sample conveying hose (105); a piezoelectric sensor (103) is arranged on the Y-shaped three-way pipe (101), and the piezoelectric sensor (103) is electrically connected with the portable signal generator (14);
the optical detection assembly (3) of the smart phone comprises a mounting base, wherein an object stage (301) and an optical detection support frame (37) are arranged on the mounting base, an LED lamp light source is arranged on the end wall of the object stage (301), an exciting light filter (33) and a light guide plate (35) are arranged on an emitting circuit of the LED lamp light source, the LED lamp light source is arranged in a socket on the side wall of the object stage (301), and the light guide plate (35) is arranged in the object stage (301); the optical detection support frame (37) is provided with an optical detection seat, and a lens (36) and an emission light filter (34) are arranged on the optical detection seat;
the optical detection seat is arranged above the object stage (301).
2. The device for absolutely quantitatively detecting the LAMP nucleic acid with multiple liquid drops according to claim 1, wherein the temperature-controlled heating platform (2) comprises a PTC electric heating plate (22), a temperature measuring hole (202) and two power interfaces (203) are arranged on the side wall of the PTC electric heating plate (22), and the temperature measuring hole (202) is electrically connected with the digital display temperature controller (21).
3. The device for absolutely quantitatively detecting the LAMP nucleic acid with multiple liquid drops according to claim 2, wherein the PTC heating plate is fixed on the detection base (4) through heat-insulating fixing plates (201) at two sides.
4. The device for absolutely quantitatively detecting the LAMP nucleic acid of multiple liquid drops according to claim 1, wherein the vertical support frame (401) is an upper and lower telescopic frame, a mounting seat (405) which moves up and down is arranged on the vertical support frame (401), the horizontal support frame (402) is inserted into the mounting seat (405), a support rod nesting hole (407) is formed in the rotary piezoelectric receptor fixing platform (403), and the rotary piezoelectric receptor fixing platform (403) is nested on the horizontal support frame (402) through the support rod nesting hole (407); the rotary piezoelectric sensor fixing platform is characterized in that columns are arranged on two sides of the top surface of the rotary piezoelectric sensor fixing platform (403) at intervals, and raised sample conveying hose placing holes (406) are formed in the mounting columns.
5. The device for absolutely quantitatively detecting the LAMP nucleic acid with multiple liquid drops according to claim 1, wherein a plurality of threaded holes are arranged on the detection base (4).
6. A method for absolutely and quantitatively detecting multiple-volume liquid drop digital LAMP nucleic acid is characterized by comprising the following steps:
1) sample processing
After sample pretreatment, extracting and purifying nucleic acid, and mixing with dye, specific primer, part of LAMP buffer solution and ribozyme-free water to prepare liquid a; mixing enzyme and partial LAMP buffer solution to prepare liquid b;
2) production of micro-droplets
Liquid a and liquid b enter a Y-shaped three-way pipe (101) through a first sample conveying hose (104) and a second sample conveying hose (105);
placing a container filled with the oil phase (12) on the PTC electric heating plate (22), and adjusting the position of the stretching tip of the micro-pipeline (102) to immerse the stretching tip into the liquid surface of the oil phase (12);
starting a portable signal generator (14) (regulating signal frequency, output voltage and pulse frequency, converting an input electric signal into a sound wave signal by a piezoelectric sensor (103) and outputting the sound wave signal to cause the vibration of a stretching tip of a micro-pipeline (102), wherein the induced vortex forms a negative pressure zone at the tip), and enabling liquid a and liquid b to enter a Y-shaped three-way pipe (101) through a first sample conveying hose and a second sample conveying hose;
then mixing the mixture at the tip and injecting the mixture into the oil phase (12) to form monodisperse LAMP reaction micro-droplets;
3) micro-droplet isothermal amplification
Removing the vibration tip device, starting the temperature control heating platform (2), and keeping the constant temperature of 60-65 ℃ for amplification for 30-60 min;
4) photographing device
Transferring the container with the collected micro liquid drops to an object stage (301) in an optical detection assembly (3) of the smart phone, placing the smart phone (31) above the device, enabling a camera of the smart phone to be opposite to a light through hole (404), starting an LED light source (32), turning on a shooting function of the smart phone (31), rotating a thread rotating sleeve to adjust an imaging focal length, collecting bright field images of the liquid drops under the condition that no optical filter is inserted, then inserting an excitation light optical filter (33) and an emission light optical filter (34), and collecting fluorescence images of the liquid drops;
5) analyzing and calculating the properties and the quantity of the micro-droplets
The picture is processed by picture coding, filtering, edge detection and the like, the average fluorescence intensity value of the negative liquid drop under the fluorescence is identified and read as a threshold value F,
if the average fluorescence intensity value of the liquid drop is greater than F, the liquid drop is positive;
if the average fluorescence intensity value of the liquid drop is less than or equal to F, the liquid drop is a negative liquid drop;
counting the number of liquid drops under a bright field, the number of positive liquid drops under fluorescence, measuring the average diameter of the liquid drops, calculating the detection rate and carrying out binarization processing, and calculating the concentration or copy number of a sample to be detected by using the following formula according to the total number of the liquid drops, the number of the positive liquid drops and the average diameter of the liquid drops, which are counted, by utilizing a Poisson distribution principle:
wherein d is the average diameter of the droplets in μm and V is the average volume of the droplets in μm;
c is the concentration or copy number of the nucleic acid to be detected, and the unit is copies/mu L;
m is the dilution factor of the sample;
p is the number of positive droplets and the unit is one;
r is the total number of droplets and is expressed in units of units.
7. The method for absolutely quantitatively detecting the multiple-volume liquid drop digital LAMP nucleic acid according to claim 6, characterized in that in the step 2), the sample injection volume ratio of liquid a to liquid b is 1:1, the liquid a is a mixed solution of a nucleic acid sample solution to be detected, a dye reagent, 4-6 template-specific primers, a part of LAMP buffer solution and ribozyme-free water, and the liquid b is a mixed solution of DNA polymerase with strand displacement activity and a part of LAMP buffer solution.
8. The method for absolutely quantitatively detecting the multiple-volume droplet digital LAMP nucleic acid according to claim 6, wherein in the step 2), the oil phase (12) is prepared by mixing the following components in a volume ratio of 100: 5-7 of mineral oil in molecular biology and surfactant.
9. The method for absolutely quantitatively detecting the LAMP nucleic acid with multiple liquid drops according to claim 6, characterized in that in the step 2), the signal frequency of the portable signal generator (13) is adjusted within the range of 85-110kHz, the output voltage is adjusted within the range of 3-20Vpp, the pulse frequency is adjusted within the range of 1-5000Hz, and the diameter of the generated micro-liquid drops is within the range of 10-600 μm.
10. Use of the multiple-volume droplet digital LAMP nucleic acid absolute quantitative detection device as defined in any one of claims 1 to 5 for detecting porcine epidemic diarrhea virus.
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| CN116497095A (en) * | 2023-01-09 | 2023-07-28 | 深圳大学 | Multiple nucleic acid quantification method, device and medium based on sediment bright field image processing |
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| CN115820816A (en) * | 2022-11-29 | 2023-03-21 | 深圳大学 | Deep learning-based multiple digital nucleic acid detection method, device and related medium |
| CN115820816B (en) * | 2022-11-29 | 2023-07-04 | 深圳大学 | Multiple digital nucleic acid detection method, device and related medium based on deep learning |
| CN116497095A (en) * | 2023-01-09 | 2023-07-28 | 深圳大学 | Multiple nucleic acid quantification method, device and medium based on sediment bright field image processing |
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