CN115992047A - Palm type integrated magnetic enrichment isothermal amplification detection equipment and microorganism detection method - Google Patents
Palm type integrated magnetic enrichment isothermal amplification detection equipment and microorganism detection method Download PDFInfo
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- CN115992047A CN115992047A CN202211206733.1A CN202211206733A CN115992047A CN 115992047 A CN115992047 A CN 115992047A CN 202211206733 A CN202211206733 A CN 202211206733A CN 115992047 A CN115992047 A CN 115992047A
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Abstract
The application provides a palm type integrated magnetism enrichment isothermal amplification detection equipment and microorganism detection method, palm type integrated magnetism enrichment isothermal amplification detection equipment includes the amplification box, the amplification frame, heating element, the light source, mixing frame and magnetic force element, the detection chamber is formed with to the amplification box, the observation hole has been seted up to the amplification box, the amplification frame is laid at the detection intracavity, the amplification frame is including the first hole of laying that is used for holding the amplification pipe, the amplification frame still includes the light-passing hole and goes out the light-passing hole, light-passing hole and go out the light-passing hole respectively with first hole intercommunication, the position and the viewing hole of light-passing hole correspond, heating element installs on the amplification frame, heating element and amplification pipe heat transfer are connected, the light source is arranged in the detection chamber, the light source is connected with the light-passing hole light path, mixing frame rotationally installs in the outside of amplification box, mixing frame includes the second hole of laying, magnetic force element installs in the outside of amplification box. The palm type integrated magnetic enrichment isothermal amplification detection equipment is convenient to carry and use on site.
Description
Technical Field
The application relates to the field of microorganism detection, in particular to palm type integrated magnetic enrichment isothermal amplification detection equipment and a microorganism detection method.
Background
Salmonella (Salmonella) is one of the important pathogens responsible for bacterial food poisoning, and is also the most complex genus of Enterobacteriaceae, belonging to the gram-negative genus. Various serotypes of salmonella can cause poisoning, most commonly salmonella typhimurium, salmonella choleraesuis, and salmonella enteritidis. Salmonella poisoning mainly has symptoms of acute gastroenteritis, latency is generally four to forty-eight hours, short term is several hours, long term is two to three days, early symptoms include nausea, headache, debilitation, coldness and the like, main symptoms include vomiting, diarrhea, abdominal pain, feces in yellow green water sample, sometimes with purulent blood and mucus, general fever temperature is between thirty-eight ℃ and forty ℃, and severe patients have symptoms of chills, convulsions and coma. The traditional PCR detection method needs the support of a PCR instrument, can only be carried out in a laboratory, and is difficult to detect timely and quickly.
The Loop-mediated isothermal amplification (LAMP-mediated isothermal amplification) is a nucleic acid amplification technology capable of starting an amplification reaction at 60-70 ℃, does not depend on a thermal cycling instrument during amplification, and is very suitable for developing corresponding miniaturized field detection equipment. In the prior art, detection devices based on LAMP appear, so that the on-site detection of salmonella is realized.
However, the samples obtained on site may contain many impurities, which may affect sensitivity of salmonella detection, and in addition, the current common LAMP detection device determines detection results by naked eye observation after amplification color development, which also easily affects detection accuracy.
Disclosure of Invention
The present application aims to solve at least one of the technical problems existing in the prior art. Therefore, the application provides palm type integrated magnetic enrichment isothermal amplification detection equipment and a microorganism detection method, and the palm type integrated magnetic enrichment isothermal amplification detection equipment can improve the accuracy of on-site detection.
According to the palm-type integrated magnetic enrichment isothermal amplification detection equipment provided by the application, the device comprises an amplification box, an amplification frame, a heating element, a light source, a mixing frame and a magnetic element, wherein a detection cavity is formed in the amplification box, an observation port communicated with the detection cavity is formed in the amplification box, a terminal is arranged on the outer side of the observation port and used for acquiring and processing fluorescent images, the amplification frame is placed in the detection cavity, the amplification frame comprises a first placing hole for containing an amplification tube, the amplification frame further comprises a light passing hole and a light outlet hole, the light passing hole and the light outlet hole are respectively communicated with the first placing hole, the position of the light outlet hole corresponds to the observation port, the heating element is installed on the amplification frame, the heating element is in heat transfer connection with the amplification tube, the light source is arranged in the detection cavity and is in light path connection with the light passing hole, the light source is used for exciting the fluorescent images, the mixing frame is rotatably installed on the outer side of the amplification box, the mixing frame comprises a second placing hole and a magnetic element, and the magnetic element is installed on the outer side of the mixing frame.
According to the palm type integrated magnetic enrichment isothermal amplification detection equipment provided by the application, the detection equipment has at least the following technical effects: through setting up mixing frame and magnetic force element, palm formula integration magnetism enrichment isothermal amplification check out test set can use the magnetic bead to carry out enrichment and elution to the sample that the scene was gathered, purification target DNA, later the rethread amplification frame carries out the amplification, carries out more accurate analysis judgement to the fluorescence image that the light source arouses through the terminal, thereby make palm formula integration magnetism enrichment isothermal amplification check out test set can improve the degree of accuracy of scene detection, palm formula integration magnetism enrichment isothermal amplification check out test set is with magnetism enrichment, LAMP and the required structure integration of fluorescence excitation on the amplification box, portable and on-the-spot use.
According to some embodiments of the application, the light source adopts the LED lamp, the central wavelength of LED lamp is 495nm, palm formula integration magnetism enrichment isothermal amplification check out test set includes first light filter and second light filter, first light filter setting is in the light source with between the light passing hole, first light filter is the band-pass filter, the central wavelength of first light filter is 498nm, the second light filter setting is in the viewing aperture, the second light filter is the high-pass filter, the initial wavelength of second light filter is 510nm.
According to some embodiments of the application, the detection chamber comprises a first chamber and a second chamber, the second chamber is located below the first chamber, the second chamber is communicated with the first chamber through a communication hole, the light source is installed in the second chamber, the amplification frame is located above the communication hole, and the light passing hole is located at the bottom of the amplification frame.
According to some embodiments of the present application, the light-passing hole is along a vertical direction, and the light-exiting hole is along a horizontal direction.
According to some embodiments of the application, the surfaces of the amplification stage and the detection chamber are light absorbing surfaces.
According to some embodiments of the application, the amplification frame is made of a metal material, the heating element is attached to the outer surface of the amplification frame, and the shape of the first mounting hole corresponds to the shape of the amplification tube.
According to some embodiments of the application, a thermally conductive silicone and/or a thermally conductive tape is applied between the heating element and the amplification stage.
According to some embodiments of the present application, the viewing port is located at the top of the amplification box, and the palm-type integrated magnetic enrichment isothermal amplification detection device includes a reflecting mirror, where the reflecting mirror is located on an optical path from the light exit hole to the viewing port.
According to the microorganism detection method provided by the application, the terminal and the palm-type integrated magnetic enrichment isothermal amplification detection equipment are used, and the microorganism detection method comprises the following steps:
s1, preparing magnetic beads coupled with the aptamer;
s2, adding a sample to be detected to separate and elute target DNA;
s3, amplifying the target DNA based on LAMP;
s4, data acquisition and processing.
According to some embodiments of the application, the sequence of the aptamer is shown as SEQ ID NO. 7.
According to some embodiments of the application, the aptamer is used at a concentration of 1-100 μm.
According to some embodiments of the application, the 5' end of the aptamer is modified with biotin.
According to some embodiments of the present application, the magnetic beads are magnetic nanoparticles surface-modified with streptavidin or avidin; preferably, the magnetic nanoparticles are polystyrene magnetic nanoparticles with streptavidin modified on the surfaces; preferably, the magnetic nanoparticles have a particle size of 300-1000nm.
According to some embodiments of the present application, the reaction conditions of step S1 include: the temperature is 24-30 ℃ and the time is 1-2h.
According to some embodiments of the present application, the weight ratio of the aptamer to the magnetic bead is 1: (1-2), preferably 1:1.
according to some embodiments of the application, the step S2 includes:
adding a sample solution to be detected and the magnetic beads coupled with the aptamer into a centrifuge tube, placing the centrifuge tube into a mixing rack, and starting the mixing rack to uniformly mix the mixed solution in the centrifuge tube so as to collect captured microorganisms;
placing the centrifuge tube near a magnetic element, and magnetically separating the magnetic beads and the mixed solution;
heating in water bath to obtain the target DNA, and transferring the target DNA to an amplification tube.
According to some embodiments of the present application, the reaction conditions for collecting captured microorganisms in step S2 include: the temperature is 64-66 ℃ and the time is 0.5-1h.
According to some embodiments of the present application, the water bath conditions in step S2 include: the temperature is 95 ℃ and the time is 5-10 min.
According to some embodiments of the application, the sample to be tested is a food, pharmaceutical or environmental sample containing microorganisms.
According to some embodiments of the present application, the microorganism comprises a food-borne pathogenic bacterium; preferably, the food-borne pathogenic bacteria comprise salmonella typhimurium.
According to some embodiments of the application, the step S3 includes:
adding an LAMP reaction system to the amplification tube, wherein the LAMP reaction system comprises a primer probe composition corresponding to the microorganism;
and heating the amplification tube by a heating element to perform LAMP reaction, thereby obtaining an LAMP reaction product.
According to some embodiments of the present application, the primer probe composition comprises:
primer:
forward outer primer F3: the sequence is shown as SEQ ID NO.2;
reverse outer primer B3: the sequence is shown as SEQ ID NO. 3;
forward inner primer FIP: the sequence is shown as SEQ ID NO. 4;
reverse inner primer BIP: the sequence is shown as SEQ ID NO. 5;
forward loop primer LF: the sequence is shown as SEQ ID NO. 6;
reverse loop primer LB: the sequence is shown as SEQ ID NO. 7;
and (3) probe: the sequence is shown as SEQ ID NO. 8.
According to some embodiments of the application, the molar ratio of F3, B3, FIP, BIP, LF, LB is 10mM.
According to some embodiments of the present application, the 3 'end of the probe is labeled with BHQ-1 and the 5' end is labeled with FAM.
According to some embodiments of the present application, the LAMP reaction system comprises: dNTPs 7-9mM, mgSO 24-26mM 4 0.8-1.2mM buffer solution and 9-11mM DNA polymerase.
According to some embodiments of the present application, the LAMP reaction system further comprises a positive control containing genomic DNA of the microorganism and a negative control that is a blank reaction solution.
According to some embodiments of the present application, the reaction conditions of the LAMP reaction include: the temperature is 64-66 ℃ and the time is 30-60min.
According to the microorganism detection method provided by the application, at least the following technical effects are achieved: due to the adoption of the palm-type integrated magnetic enrichment isothermal amplification detection equipment, the microorganism detection method is suitable for field operation, microorganisms in samples can be detected timely and rapidly, the acquired samples are magnetically enriched before LAMP reaction, target DNA is purified, the effect of LAMP reaction is improved, the fluorescent images are further accurately analyzed and judged through the terminal, the detection result is accurately estimated, and the accuracy of field detection is improved by the microorganism detection method.
According to some embodiments of the application, the step S4 includes:
a light source excites the LAMP reaction product;
the terminal obtains fluorescent images of the amplification tubes of the control group and the experimental group;
calculating a detection threshold based on the fluorescence images of the control group, the detection threshold being equal to
Calculating a signal value based on said fluorescence images of said experimental group, said signal value being equal to +.>
Wherein R, G, B is the average fluorescence intensity of R channel, G channel and B channel, and sigma is the standard deviation of the fluorescence intensity of G channel of the control group;
and comparing the detection threshold value with the signal value to obtain a detection result of the experiment group, and visually displaying the signal value, the detection threshold value and the detection result.
According to some embodiments of the application, the step S4 includes: and exciting the LAMP reaction product by a light source, observing the fluorescence change of the amplification tube by naked eyes, and if green fluorescence appears, determining that the sample to be detected contains the microorganism.
Drawings
The foregoing and/or additional aspects and advantages of the present application will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings, wherein:
FIG. 1 is a schematic axial view of a palm-type integrated magnetic enrichment isothermal amplification detection device according to an embodiment of the present application;
FIG. 2 is an exploded schematic view of a palm-type integrated magnetic enrichment isothermal amplification detection device according to an embodiment of the present application;
FIG. 3 is a schematic cross-sectional view of a palm-type integrated magnetic enrichment isothermal amplification detection device according to an embodiment of the present application;
FIG. 4 is a schematic cross-sectional view of an amplification stage according to an embodiment of the present application;
FIG. 5 is a schematic diagram of a graphical user interface of a terminal as used herein;
FIG. 6 is a schematic diagram of another graphical user interface of a terminal as used herein;
FIG. 7 is a specific experiment of LAMP primer compositions of Salmonella typhimurium in the present application;
FIG. 8 is a flow chart of a method of sensitivity experiment of the LAMP primer composition of Salmonella typhimurium of the present application;
FIG. 9 is a sensitivity experiment of LAMP primer compositions of Salmonella typhimurium of the present application;
FIG. 10 is an effect comparison of LAMP method and magnetic separation LAMP method for detecting Salmonella typhimurium; FIGS. 10a and 10c show the sensitivity of the LAMP method; FIGS. 10b and 10d show the sensitivity of the magnetic separation LAMP method;
FIG. 11 compares the effect of magnetic separation LAMP with that of ordinary LAMP using actual samples.
Reference numerals:
the upper case 110, the viewing port 111, the second optical filter 112, the middle case 120, the mirror 121, the first optical filter 122, the lower case 130, the switch 131, the knob 132, the first chamber 140, the second chamber 150, the third chamber 160, the amplification stage 200, the first mounting hole 210, the light passing hole 220, the light exiting hole 230, the heating element 300, the motor 400, the mixing stage 500, the magnetic element 600, the amplification stage 710, the terminal 900, the first display region 911, the second display region 912, the third display region 913, the first interaction region 921, the second interaction region 922, the third interaction region 923, the fourth interaction region 924, the fourth display region 931, the fifth display region 932, and the sixth display region 933.
Detailed Description
Embodiments of the present application are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are exemplary only for the purpose of explaining the present application and are not to be construed as limiting the present application.
In the description of the present application, it should be understood that references to orientation descriptions, such as directions of up, down, front, back, left, right, etc., are based on the orientation or positional relationship shown in the drawings, are merely for convenience of describing the present application and simplifying the description, and do not indicate or imply that the apparatus or element referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present application.
In the description of the present application, the meaning of a number is one or more, the meaning of a number is two or more, greater than, less than, exceeding, etc. are understood to not include the present number, and the meaning of a number above, below, within, etc. are understood to include the present number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present application, unless explicitly defined otherwise, terms such as arrangement, installation, connection, etc. should be construed broadly and the specific meaning of the terms in the present application can be reasonably determined by a person skilled in the art in combination with the specific contents of the technical solution.
Referring to fig. 1 and 2, according to the palm-type integrated magnetic enrichment isothermal amplification detection device provided by the application, the device comprises an amplification box, an amplification frame 200, a heating element 300, a light source, a mixing frame 500 and a magnetic element 600, wherein the amplification box is provided with a detection cavity, an observation port 111 communicated with the detection cavity is formed in the amplification box, a terminal 900 is arranged outside the observation port 111 and used for acquiring and processing fluorescent images, the amplification frame 200 is placed in the detection cavity, the amplification frame 200 comprises a first placing hole 210 used for containing an amplification tube 710, the amplification frame 200 further comprises a light through hole 220 and a light outlet hole 230, the light through hole 220 and the light outlet hole 230 are respectively communicated with the first placing hole 210, the position of the light outlet hole 230 corresponds to the observation port 111, the heating element 300 is installed on the amplification frame 200, the heating element 300 is in heat transfer connection with the amplification tube 710, the light source is arranged in the detection cavity and in light path connection with the light through hole 220, the light source is used for exciting fluorescent images, the mixing frame 500 is rotatably installed outside the amplification box, the mixing frame 500 comprises a second placing hole used for containing a centrifuge tube, and the magnetic element 600 is installed outside the amplification box.
When in use, firstly, the collected sample is purified through the mixing rack 500 and the magnetic force element 600, then the target DNA is amplified through the amplifying rack 200 and the heating element 300, finally, fluorescence is excited through the light source, a fluorescence image is collected through the terminal 900, and the detection result is analyzed.
According to the palm-type integrated magnetic enrichment isothermal amplification detection device, through setting up mixing frame 500 and magnetic element 600, palm-type integrated magnetic enrichment isothermal amplification detection device can use magnetic beads to carry out enrichment and elution to the sample that on-the-spot was gathered, purification target DNA, later rethread amplification frame 200 carries out the amplification, carry out more accurate analysis judgement through the fluorescence image that terminal 900 excited the light source, thereby make palm-type integrated magnetic enrichment isothermal amplification detection device can improve the degree of accuracy of on-the-spot detection, palm-type integrated magnetic enrichment isothermal amplification detection device is with magnetism enrichment, LAMP and the required structure integration of fluorescence excitation are on the amplification box, portable and on-the-spot use.
The terminal 900 may specifically include, but is not limited to, a smart phone, a tablet computer, a notebook computer, a desktop computer, or the like having an image capturing function. Generally, the terminal includes: a processor and a memory. The processor may be implemented in at least one hardware form of DSP (Digital Signal Processing ), FPGA (Field-Programmable Gate Array, field programmable gate array), PLA (Programmable Logic Array ). The memory is at least used for storing a computer program which, after being loaded and executed by the processor, enables the acquisition and analysis processing of the fluoroscopic image by the terminal. In addition, the resources stored in the memory can also comprise an operating system, data and the like, and the storage mode can be short-term storage or permanent storage. The operating system may include Windows, unix, linux, among others. The data may include, but is not limited to, image data, processing results, and the like. In some embodiments, the terminal may further include a display screen, an input-output interface, a communication interface, a sensor, a power supply, and a communication bus.
By "heat transfer connection" is meant that heat generated by the heating element 300 can be transferred directly or indirectly to the amplification tubes 710, thereby heating the liquid within the amplification tubes 710 and facilitating the LAMP reaction.
In this application, the quality of the fluorescent image determines the accuracy of detection to a great extent, for this reason, according to some embodiments of the present application, the light source adopts the LED lamp, the central wavelength of the LED lamp is 495nm, the handheld integrated magnetic enrichment isothermal amplification detection device includes the first light filter 122, the first light filter 122 sets up between light source and the light through hole 220, the first light filter 122 is the band-pass filter, the central wavelength of the first light filter 122 is 498nm. The first optical filter 122 can filter stray light emitted by the light source, so as to perform targeted irradiation on the target DNA, and improve the quality of fluorescence excitation.
Further, the palm-type integrated magnetic enrichment isothermal amplification detection device comprises a second optical filter 112, the second optical filter 112 is arranged at the observation port 111, the second optical filter 112 is a high-pass optical filter, and the initial wavelength of the second optical filter 112 is 510nm. The second optical filter 112 can filter out the background stray light existing in the detection cavity, so as to further improve the quality of the fluorescent image acquired by the terminal 900.
To reduce background glare from the source, in some embodiments, referring to fig. 3, the detection chamber includes a first chamber 140 and a second chamber 150, the second chamber 150 is located below the first chamber 140, the second chamber 150 and the first chamber 140 are communicated through a communication hole, a light source is installed in the second chamber 150, the amplification stage 200 is located above the communication hole, and a light passing hole 220 is located at the bottom of the amplification stage 200. By designing the first chamber 140 and the second chamber 150 to be separated, the amplification frame 200 covers the communication hole, so that light emitted by the light source can only be emitted to the amplification tube 710 from the communication hole and the light passing hole 220 without leaking to other parts of the first chamber 140, and interference of the light source on acquisition of fluorescent images is avoided.
In some embodiments, referring to fig. 4, the light-passing holes 220 are in a vertical direction and the light-exiting holes 230 are in a horizontal direction. The axes of the light-passing hole 220 and the light-emitting hole 230 are perpendicular to each other, so that the light emitted from the light-passing hole 220 cannot directly enter the light-emitting hole 230, and interference of stray light on fluorescent image acquisition is avoided.
In some embodiments, the surfaces of the amplification stage 200 and detection chamber are light absorbing surfaces. The light absorption surface can weaken or even eliminate reflection, and interference of stray light on acquisition of fluorescent images is avoided.
In this application, the quality of the LAMP reaction also affects the final detection quality, and for this purpose, it is necessary to precisely control the heating temperature of the amplification tube 710. According to some embodiments of the present application, the amplification stage 200 is made of a metal material, the heating element 300 is attached to the outer surface of the amplification stage 200, the shape of the first receiving hole 210 corresponds to the shape of the amplification tube 710, and the inner wall of the first receiving hole 210 contacts the amplification tube 710. On the one hand, the heating element 300 does not directly contact with the amplification tube 710, but conducts heat to the amplification tube 710 through the amplification frame 200, and on the other hand, the shape of the first mounting hole 210 corresponds to the amplification tube 710, so that the contact surface between the amplification frame 200 and the amplification tube 710 is increased, the heating uniformity is further improved, and the temperature difference of liquid at different parts in the amplification tube 710 is reduced.
In some embodiments, a thermally conductive silicone and/or a thermally conductive tape is applied between the heating element 300 and the amplification stage 200. Both the heat conductive silicone grease and the heat conductive tape can fill up the irregularities or voids of the surfaces of the heating element 300 and the amplification stage 200, so that the heat conduction efficiency between the heating element 300 and the amplification stage 200 is higher.
The heating element 300 may be a PTC ceramic heating plate with a control circuit, or other heating scheme with temperature regulation.
According to some embodiments of the present application, the viewing port 111 is located at the top of the amplification cassette, and the palm-type integrated magnetic enrichment isothermal amplification detection device includes a mirror 121, where the mirror 121 is located on the optical path from the light exit aperture 230 to the viewing port 111. The top of the amplification cassette forms a platform on which the terminal 900 can be conveniently placed to capture fluorescent images, eliminating the need for additional fixing structures. The optical path is adjusted by the mirror 121, so that fluorescence is accurately emitted from the observation port 111.
According to the microorganism detection method provided by the application, the palm type integrated magnetic enrichment isothermal amplification detection equipment provided by the application is used, and the biological detection method comprises the following steps of:
s1, preparing magnetic beads coupled with the aptamer;
s2, adding a sample to be detected to separate and elute target DNA;
s3, amplifying target DNA based on LAMP;
s4, data acquisition and processing.
According to some embodiments of the present application, the sequence of the aptamer is shown as SEQ ID NO. 7.
According to some embodiments of the application, the aptamer is used at a concentration of 1-100 μm.
According to some embodiments of the present application, the 5' end of the aptamer is modified with biotin.
According to some embodiments of the present application, the magnetic beads are magnetic nanoparticles surface-modified with streptavidin or avidin; preferably, the magnetic nanoparticles are polystyrene magnetic nanoparticles with streptavidin-modified surfaces; preferably, the magnetic nanoparticles have a particle size of 300-1000nm.
According to some embodiments of the present application, the reaction conditions of step S1 include: the temperature is 24-30 ℃ and the time is 1-2h.
According to some embodiments of the present application, the weight ratio of the aptamer to the magnetic bead is 1: (1-2), preferably 1:1.
in some embodiments, step S2 comprises:
adding a sample solution and magnetic beads into a centrifuge tube, placing the centrifuge tube into a mixing rack 500, and starting the mixing rack 500 to uniformly mix the mixed solution in the centrifuge tube so as to collect captured microorganisms;
placing a centrifuge tube near the magnetic element 600, magnetically separating the magnetic beads and the mixed solution;
the target DNA is obtained by heating in a water bath, and transferred to the amplification tube 710.
According to some embodiments of the present application, the reaction conditions for collecting captured microorganisms in step S2 include: the temperature is 64-66 ℃ and the time is 0.5-1h.
According to some embodiments of the present application, the water bath conditions in step S2 include: the temperature is 95 ℃ and the time is 5-10 min.
According to some embodiments of the present application, the microorganism comprises a food-borne pathogenic bacterium; preferably, the food-borne pathogenic bacteria comprise salmonella typhimurium.
According to some embodiments of the present application, the sample to be tested is a food, pharmaceutical or environmental sample containing microorganisms.
Due to the adoption of the palm-type integrated magnetic enrichment isothermal amplification detection equipment, the microorganism detection method is suitable for field operation, is convenient to timely and rapidly detect microorganisms in a sample, and can be used for purifying target DNA by carrying out magnetic enrichment on the collected sample before LAMP reaction, so that the effect of LAMP reaction is improved, further, a fluorescent image is accurately analyzed and judged through a terminal, so that a detection result is more accurately evaluated, and the accuracy of field detection is improved.
In some embodiments, step S3 comprises:
adding an LAMP reaction system to the amplification tube, wherein the LAMP reaction system comprises a primer probe composition corresponding to the microorganism;
the heating element heats the amplification tube to perform LAMP reaction, and LAMP reaction products are obtained.
According to some embodiments of the present application, a primer probe composition includes:
primer:
forward outer primer F3: the sequence is shown as SEQ ID NO.2;
reverse outer primer B3: the sequence is shown as SEQ ID NO. 3;
forward inner primer FIP: the sequence is shown as SEQ ID NO. 4;
reverse inner primer BIP: the sequence is shown as SEQ ID NO. 5;
forward loop primer LF: the sequence is shown as SEQ ID NO. 6;
reverse loop primer LB: the sequence is shown as SEQ ID NO. 7;
and (3) probe: the sequence is shown as SEQ ID NO. 8.
According to some embodiments of the present application, the molar ratio of F3, B3, FIP, BIP, LF, LB is 10mM.
According to some embodiments of the present application, the 3 'end of the probe is labeled with BHQ-1 and the 5' end is labeled with FAM.
According to some embodiments of the present application, the LAMP reaction system comprises: dNTPs 7-9mM, mgSO 24-26mM 4 0.8-1.2mM buffer solution and 9-11mM DNA polymerase.
It will be appreciated that the unit mM is a shorthand for mmol/L.
According to some embodiments of the present application, the LAMP reaction system further comprises a positive control and a negative control, wherein the positive control contains genomic DNA of the microorganism, and the negative control is blank reaction solution, i.e. does not contain the bacteria to be tested and the templates of the bacteria to be tested.
According to some embodiments of the present application, the reaction conditions of the LAMP reaction include: the temperature is 64-66 ℃ and the time is 30-60min.
In some embodiments, step S4 comprises:
the light source excites LAMP reaction products;
the terminal 900 acquires fluorescence images of the amplification tubes 710 of the control and experimental groups;
calculating a detection threshold based on the fluorescence image of the control group, the detection threshold being equal to
Calculating a signal value based on the fluorescent images of the experimental group, the signal value being equal to +.>
Wherein R, G, B is the average fluorescence intensity of R channel, G channel and B channel, and sigma is the standard deviation of the fluorescence intensity of G channel of the control group;
and comparing the detection threshold value with the signal value to obtain a detection result of the experiment group, and visually displaying the signal value, the detection threshold value and the detection result.
In some embodiments, step S4 comprises: the light source excites the LAMP reaction product, and the fluorescence change of the amplification tube 710 is observed by naked eyes, if green fluorescence appears, the sample to be detected contains microorganisms.
Fig. 5 is a graphical user interface for displaying results of the terminal 900, showing an embodiment of a visual display of the terminal 900, and fig. 5 includes a first display area 911, a second display area 912 and a third display area 913, where the first display area 911 displays fluorescent images of a control group, the second display area 912 displays fluorescent images of each experimental group, and corresponding detection results are marked under the images, and the third display area 913 displays signal values of each experimental group in the form of a bar graph and shows detection threshold values in dashed lines. The acquired fluorescent images of the control group and the experimental group are respectively displayed, and the signal value and the detection threshold value of each experimental group are represented through a bar graph, so that the detection result is clearly and intuitively known.
Fig. 6 is a data processing graphical user interface of the terminal 900, and fig. 6 includes a first interaction zone 921, a second interaction zone 922, a third interaction zone 923, and a fourth interaction zone 924. Clicking the first interaction area 921, and selecting a fluorescence image for detection and analysis at the time, wherein the fluorescence image is displayed in a fourth display area 931; then clicking the second interaction area 922, further selecting a fluorescence image serving as a control group from the fluorescence images, wherein the fluorescence image of the control group is displayed in the fifth display area 932; then clicking the third interaction area 923 to select a fluorescent image serving as an experiment group, wherein the fluorescent image of the experiment group is displayed in a sixth display area 933; finally, the fourth interactive area 924 is clicked, and the terminal 900 calculates the relevant value by the processor and automatically jumps to the result display graphical user interface.
The palm-type integrated magnetic enrichment isothermal amplification detection device and the microorganism detection method provided according to the present application are described in detail below with reference to fig. 1 to 5 in a specific embodiment. It is to be understood that the following description is exemplary only and is not intended to limit the application to the details of the present application. The present embodiment may also be replaced by or combined with the above-described corresponding technical features.
The palm type integrated magnetic enrichment isothermal amplification detection device comprises an amplification box, wherein the amplification box comprises an upper box body 110, a middle box body 120 and a lower box body 130, the middle box body 120 is arranged on the lower box body 130, and the upper box body 110 is arranged on the middle box body 120. The upper case 110, the middle case 120, and the lower case 130 enclose a detection chamber, the middle case 120 partitions the detection chamber into a first chamber 140 and a second chamber 150, and the first chamber 140 and the second chamber 150 are communicated through communication holes on the middle case 120. The upper case 110 can be separated from the middle case 120 to expose the first chamber 140. A third chamber 160 is also formed between the middle case 120 and the lower case 130, and a power module and a control module (not shown) are installed in the third chamber 160.
The palm type integrated magnetic enrichment isothermal amplification detection device comprises a knob 132, a motor 400, a mixing rack 500 and a magnetic element 600. The motor 400 is installed at a side of the upper case 110, the mixing rack 500 is installed on the motor 400, the mixing rack 500 includes a second receiving hole for receiving a centrifuge tube (EP tube), and the magnetic member 600 is installed at the top of the upper case 110. The knob 132 is mounted on the lower case 130, and the knob 132, the power module, the control module, and the motor 400 are connected in communication.
The palm-type integrated magnetic enrichment isothermal amplification detection device comprises a switch 131, an amplification frame 200 and a heating element 300, wherein the amplification frame 200 is made of aluminum alloy, the surface of the amplification frame 200 is oxidized and processed to be black, the amplification frame 200 is provided with a first placing hole 210, a light passing hole 220 and a light emitting hole 230 along the vertical direction, the light passing hole 220 and the light emitting hole 230 are respectively communicated with the first placing hole 210, the light passing hole 220 is positioned at the bottom of the amplification frame 200, and the shape of the first placing hole 210 corresponds to that of an amplification tube 710. The heating element 300 is attached to the outer surface of the amplification stage 200, and a heat conductive silicone grease and a heat conductive adhesive tape are coated between the heating element 300 and the amplification stage 200. The heating element 300 adopts PTC ceramic heating plates, the switch 131 is arranged on the lower box body 130, and the switch 131, the power supply module, the control module and the heating element 300 are in communication connection.
The palm type integrated magnetic enrichment isothermal amplification detection device comprises a light source, a reflector 121, a first optical filter 122 and a second optical filter 112, wherein the light source adopts an LED lamp, the central wavelength of the LED lamp is 495nm, the light source is arranged in a second cavity 150, the first optical filter 122 is arranged between the light source and a light passing hole 220, the first optical filter 122 is a band-pass optical filter, and the central wavelength of the first optical filter 122 is 498nm. The amplification stage 200 is placed above the communication hole corresponding to the light passing hole 220. The top of the upper case 110 is provided with an observation port 111, the second optical filter 112 is disposed at the observation port 111, the second optical filter 112 is a high-pass optical filter, and the initial wavelength of the second optical filter 112 is 510nm. The middle case 120 is made of temperature-resistant black plastic, the middle case 120 forms an inclined mounting table, and the reflector 121 is mounted on the mounting table, so that light emitted from the light emitting hole 230 is reflected to the viewing port 111.
The method for detecting the microorganisms by using palm type integrated magnetic enrichment isothermal amplification detection equipment comprises the following steps:
s1, preparing magnetic beads coupled with the aptamer;
s2, adding a sample to be detected to separate and elute target DNA;
s3, amplifying target DNA based on LAMP;
s4, data acquisition and processing.
The step S2 comprises the following steps:
adding a sample solution and magnetic beads coupled with an aptamer into a centrifuge tube, placing the centrifuge tube into a mixing rack 500, and starting the mixing rack 500 to mix the mixed solution in the centrifuge tube uniformly;
placing a centrifuge tube near the magnetic element 600, magnetically separating the magnetic beads and the mixed solution;
the target DNA is obtained by heating in a water bath, and transferred to the amplification tube 710.
The step S3 comprises the following steps:
adding an LAMP reaction system to the amplification tube, wherein the LAMP reaction system comprises a primer composition corresponding to the microorganism;
the heating element heats the amplification tube to perform LAMP reaction, and LAMP reaction products are obtained.
In some embodiments, step S5 comprises:
the light source excites LAMP reaction products;
the terminal 900 acquires fluorescence images of the amplification tubes 710 of the control and experimental groups;
calculating a detection threshold based on the fluorescence image of the control group, the detection threshold being equal to
Calculating a signal value based on the fluorescent images of the experimental group, the signal value being equal to +.>
Wherein R, G, B is the average fluorescence intensity of R channel, G channel and B channel, and sigma is the standard deviation of the fluorescence intensity of G channel of the control group;
and comparing the detection threshold value with the signal value to obtain a detection result of the experiment group, and visually displaying the signal value, the detection threshold value and the detection result.
Other embodiments to which this application relates are described below:
example 1
Specific experiments with LAMP primer compositions for detection of Salmonella typhimurium.
1. Preparing gene templates of Salmonella typhimurium, staphylococcus aureus, shigella, listeria monocytogenes and campylobacter as a control group for a specific experiment;
2. preparation of Salmonella typhimurium specific primers (synthesized by Shanghai Biotechnology Co., ltd.), salmonella typhimurium specific probes (synthesized by Shanghai Biotechnology Co., ltd.), bst DNA polymerase (New England Biolabs, inc.), dNTP (Beijing full gold Biotechnology Co., ltd.), LAMP buffer solution (Beijing full gold Biotechnology Co., ltd.), mgSO 4 (Sigma-Aldrich Chemical co. (St.Louis, MO, USA)), SYTO9 nucleic acid dye (Invitrogen co. (Carlsbad, CA, USA)), ultrapure water;
3. the LAMP reaction system (unit: μL) was configured in the following volume:
LAMP buffer solution: 2.5,0.8-1.2mM;
MgSO 4 ：1.5，24-26mM；
dNTPs：4，7-9mM；
FIP BIP：4，10mM；
LOOP：2，10mM；
F3 B3：0.5，10mM；
BST：1，9-11mM；
syto9 (or specific probe): 0.25 10mM;
a DNA template: 2.5
Ultrapure water: 6.75;
4. the LAMP reaction system was prepared, and then isothermal nucleic acid amplification was quantitatively detected at 64.7℃for 1 hour using a CFX96 real-time PCR apparatus (Bio-Rad Laboratories Inc. (Hercules, calif., USA)), and the amplification curve was observed.
The primer probe sets are shown in Table 1.
TABLE 1
The experimental results are shown in fig. 7, the salmonella typhimurium peaks at about 17 minutes, while other pathogenic templates and no template controls do not. The results show that the primer set is specific to Salmonella typhimurium only.
Example 2
Sensitivity experiments of LAMP primer compositions for detection of Salmonella typhimurium. The flow chart of the detection method is shown in fig. 8.
1. The salmonella typhimurium is selected into 10mL of LB broth, and is cultured for 8 to 12 hours in a shaking table with constant temperature of 37 ℃;
2. taking 1mL of cultured salmonella typhimurium, putting the salmonella typhimurium into a centrifuge, centrifuging at a high speed (10000 g) for 5 minutes, removing supernatant, re-dissolving the precipitate by using 1mL of 1 XPBS solution, and shaking uniformly;
3. diluting the bacterial solution by 10 times of gradient by using 1 XPBS solution, coating the bacterial solution of each gradient on a Maiconkai agar medium for plate counting, and placing the bacterial solution into a refrigerator at 4 ℃ for later use;
4. simultaneously, 100. Mu.M of an aptamer solution (synthesized by Shanghai Biotechnology Co., ltd., 5'biotin-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3' (SEQ ID NO. 1)) was prepared and reacted with an equivalent amount of streptavidin polystyrene magnetic microspheres (Tianjin Sile technology research center, 300nm,5 mg/mL) for 45 minutes;
5. biotin aptamer was reacted with streptavidin polystyrene magnetic microspheres and washed 2 times on a magnetic separation rack using PBST solution (1 x Phosphate Buffer Saline (PBS) containing 0.05% tween 20 (PBST));
6. sealing the streptavidin polystyrene magnetic microsphere for 15 minutes by using equivalent sealing liquid (salcon sperm DNA 1mg/mL and yeast tRNA 0.1 mg/mL), washing twice by using PBST solution, and adding equivalent PBS solution for redissolution;
7. mixing 100 mu L of bacterial liquid with each gradient with 25 mu L of magnetic bead solution respectively, and reacting for 1-2 hours;
8. removing the supernatant by using a magnetic separator, adding 40 mu L of pure water for redissolution, putting into a water bath kettle at 95 ℃ for 5 minutes for cell lysis, and then putting into ice for 5 minutes for DNA precipitation to obtain a template to be detected;
9. then, the nucleic acid amplification step of example 1 was performed to obtain an amplification curve, and the colony count result (number of bacteria) obtained in step 3 was one-to-one correlated with the experimental group to obtain a final sensitivity result.
The results showed that the LAMP method (FIG. 9) had a good linear relationship, the linear relationship was >95%, and that the LAMP method could detect 5.5CFU/mL of Salmonella typhimurium, exceeding most nucleic acid detection methods.
Comparative example 1
Salmonella typhimurium was detected by LAMP alone without magnetic enrichment of the appropriate ligand (or binding to other aptamers), and the results (target gene concentration, purity, detection sensitivity) were compared with the examples. The LAMP method is shown on the left, and the magnetic separation LAMP method is shown on the right.
1. The salmonella typhimurium is selected into 10mL of LB broth, and is cultured for 8 to 12 hours in a shaking table with constant temperature of 37 ℃;
2. taking 1mL of cultured salmonella typhimurium, putting the salmonella typhimurium into a centrifuge, centrifuging at a high speed (10000 g) for 5 minutes, removing supernatant, re-dissolving the precipitate by using 1mL of 1 XPBS solution, and shaking uniformly;
3. bacterial solutions were 10-fold diluted with 1 XPBS solution and plating plates were counted for each gradient on MAIKAI agar medium;
4. and (3) putting the bacterial liquid into a water bath kettle at 95 ℃ for 5 minutes to perform cell lysis, then putting into ice for 5 minutes to perform DNA precipitation to obtain a template to be detected, then performing nucleic acid amplification, and observing an amplification curve.
The results showed that the conventional LAMP method was only able to detect 550CFU/mL bacteria and had a poor linear relationship (FIG. 10a & FIG. 10 c), whereas the magnetic separation LAMP was able to detect 5.5CFU/mL, which was two orders of magnitude (100-fold) more sensitive than the conventional method, and had a good linear relationship (R2 > 95%) (FIGS. 10b & 10 d).
Comparative example 2
The effect of magnetic separation LAMP was compared with that of ordinary LAMP using 10g of meat lump sample, and the results are shown in FIG. 11.
The results show that the method based on magnetic separation and LAMP utilizes aptamer magnetic enrichment to obviously increase the purity and concentration of target genes, avoids the traditional pre-enrichment culture and redundant nucleic acid extraction process, has the characteristics of strong specificity, high accuracy, simple operation, low cost, difficult inactivation and the like, combines the magnetic enrichment and LAMP, and provides a novel detection technical scheme for rapidly detecting salmonella typhimurium, and the method is easy to popularize and apply on a large scale.
In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the application, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. The palm type integrated magnetic enrichment isothermal amplification detection device is characterized by comprising the following components:
the amplification box is provided with a detection cavity, an observation port communicated with the detection cavity is formed in the amplification box, a terminal is arranged on the outer side of the observation port, and the terminal is used for acquiring and processing fluorescent images;
the amplification frame is arranged in the detection cavity and comprises a first placing hole for accommodating an amplification tube, a light passing hole and a light emitting hole, wherein the light passing hole and the light emitting hole are respectively communicated with the first placing hole, and the position of the light emitting hole corresponds to the observation port;
a heating element mounted on the amplification stage, the heating element in heat transfer connection with the amplification tube; the light source is arranged in the detection cavity and is connected with the light-passing hole in a light path, and the light source is used for exciting the fluorescent image;
the mixing rack is rotatably arranged at the outer side of the amplification box and comprises a second placing hole for accommodating a centrifuge tube;
and the magnetic force element is arranged on the outer side of the amplification box.
2. The palm-type integrated magnetic enrichment isothermal amplification detection device according to claim 1, wherein: the light source adopts the LED lamp, the central wavelength of LED lamp is 495nm, palm formula integration magnetism enrichment isothermal amplification check out test set includes first light filter and second light filter, first light filter sets up the light source with between the light passing hole, first light filter is the band-pass filter, the central wavelength of first light filter is 498nm, the second light filter sets up the viewing aperture, the second light filter is the high-pass filter, the initial wavelength of second light filter is 510nm.
3. The palm-type integrated magnetic enrichment isothermal amplification detection device according to claim 2, wherein: the detection cavity comprises a first cavity and a second cavity, the second cavity is located below the first cavity, the second cavity is communicated with the first cavity through a communication hole, the light source is installed in the second cavity, the amplification frame is located above the communication hole, and the light passing hole is located at the bottom of the amplification frame.
4. The palm-type integrated magnetic enrichment isothermal amplification detection device according to claim 3, wherein: the light passing holes are along the vertical direction, and the light emitting holes are along the horizontal direction.
5. The palm-type integrated magnetic enrichment isothermal amplification detection device according to claim 3, wherein: the surfaces of the amplification frame and the detection cavity are light absorption surfaces.
6. The palm-type integrated magnetic enrichment isothermal amplification detection device according to claim 1, wherein: the amplification frame is made of metal materials, the heating element is attached to the outer surface of the amplification frame, and the shape of the first placing hole corresponds to the shape of the amplification tube.
7. The palm-type integrated magnetic enrichment isothermal amplification detection device according to claim 6, wherein: and heat conduction silicone grease and/or heat conduction adhesive tape are/is coated between the heating element and the amplification frame.
8. The palm-type integrated magnetic enrichment isothermal amplification detection device according to claim 1, wherein: the observation port is positioned at the top of the amplification box, and the palm-type integrated magnetic enrichment isothermal amplification detection device comprises a reflecting mirror, and the reflecting mirror is positioned on a light path from the light emergent hole to the observation port.
9. A method for detecting microorganisms, characterized in that the method for detecting microorganisms uses a terminal and the palm-type integrated magnetic enrichment isothermal amplification detection device according to any one of claims 1 to 8, preferably the method for detecting microorganisms comprises the steps of:
s1, preparing magnetic beads coupled with the aptamer;
s2, adding a sample to be detected to separate and elute target DNA;
s3, amplifying the target DNA based on LAMP;
s4, data acquisition and processing;
preferably, the step S3 includes:
adding an LAMP reaction system to an amplification tube, wherein the LAMP reaction system comprises a primer probe composition corresponding to microorganisms;
heating the amplification tube by a heating element to perform LAMP reaction to obtain an LAMP reaction product;
preferably, the LAMP reaction system comprises a primer probe composition;
the primer comprises:
forward outer primer F3: the sequence is shown as SEQ ID NO.2;
reverse outer primer B3: the sequence is shown as SEQ ID NO. 3;
forward inner primer FIP: the sequence is shown as SEQ ID NO. 4;
reverse inner primer BIP: the sequence is shown as SEQ ID NO. 5;
forward loop primer LF: the sequence is shown as SEQ ID NO. 6;
reverse loop primer LB: the sequence is shown as SEQ ID NO. 7;
the sequence of the probe is shown as SEQ ID NO. 8.
10. The method of claim 9, wherein the LAMP reaction system further comprises: buffer solution, DNA polymerase, magnesium ions, dNTPs; preferably, the concentration of each component in the LAMP reaction system is: 7-9mM dNTPs, 24-26mM magnesium ions, 0.8-1.2mM buffer solution and 9-11mM DNA polymerase.
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