阅读说明：本技术 一种微孔载体检测抑制物的测定方法 (Determination method for detecting inhibitor by microporous carrier ) 是由 张君成 王忠文 张正淳 于 2020-07-07 设计创作，主要内容包括：本发明公开了一种微孔载体检测抑制物的测定方法,在微孔板的微孔内注入40μl的琼脂培养基制成微孔载体,在微孔载体上载入药物样品以及病原菌孢子,检测药物对孢子萌发的抑制作用,测定方法的步骤如下：1)用微孔板制备合格的微孔载体；2)将待测样品载入微孔载体；3)将病原菌孢子载入微孔载体；4)孢子萌发培养；5)结果观测。本发明的优点：1)一块常规微孔板可以制备96个微孔载体,可以实现测试体系微型化；2)一个测试单元只消耗样品液10μl,实现检测样本微量化。(The invention discloses a method for measuring a micropore carrier detection inhibitor, which comprises the following steps of injecting 40 mul of agar culture medium into micropores of a micropore plate to prepare a micropore carrier, loading a medicine sample and pathogenic bacteria spores on the micropore carrier, and detecting the inhibition effect of the medicine on spore germination: 1) preparing a qualified microporous carrier by using a microporous plate; 2) loading a sample to be detected into a microporous carrier; 3) loading pathogenic spores into a microporous carrier; 4) spore germination and culture; 5) and (5) observing the result. The invention has the advantages that: 1) 96 micropore carriers can be prepared by one conventional micropore plate, and the miniaturization of a test system can be realized; 2) one test unit only consumes 10 mul of sample liquid, and the micro-quantification of the detection sample is realized.)

1. A method for measuring the detection inhibitor of a microporous carrier is characterized in that 40 mul of agar culture medium is injected into micropores of a microporous plate to prepare the microporous carrier, a medicine sample and pathogenic bacteria spores are loaded on the microporous carrier, and the inhibition effect of the medicine on the spore germination is detected, wherein the measuring method comprises the following steps:

1) preparation of qualified microporous carrier by microporous plate

Stably clamping a microporous plate: preparing a clamping and stabilizing tool which can clamp the microporous plate stably and flatly and has a clean surface, transferring the clamping and stabilizing tool to a sterile workbench, and clamping the clean microporous plate on the clamping and stabilizing tool;

melting of agar culture medium: heating and melting agar culture medium filled in a triangular flask and suitable for spore germination of pathogenic bacteria;

③ treatment of culture medium at 80 ℃: heating a common water bath kettle to a constant temperature of 80 ℃ in advance, putting the melted culture medium into the water bath kettle, and balancing to 80 ℃;

fourthly, the pipette gun does not have the processing of bubble pipetting: transferring the culture medium balanced to 80 ℃ to a sterile workbench, adjusting the liquid taking amount of a liquid-transferring gun to 40 mu l, and extending a gun head into the culture medium at 80 ℃ to repeatedly suck and discharge the culture medium until no bubble phenomenon occurs in liquid discharging;

preparing a microporous carrier: after the operation of no bubble phenomenon, excessively pressing a pipette gun to suck the culture medium, lifting the gun head, removing the culture medium adhered to the outside of the gun head by attaching the pipette head to the wall of the bottle mouth of the triangular flask, transferring the culture medium into the micropores of the microporous plate, injecting 40 mul of culture medium to the bottom of the micropores, keeping the gun head in the culture medium liquid for circular movement, guiding the culture medium to be uniformly distributed on the bottom surface of the micropores, and cooling to form a tiny flat plate of the agar culture medium as a micropore carrier for testing; after a micropore is injected, a mother finger presses a pipette gun, returns to and moves into a culture medium in a triangular flask at the temperature of 80 ℃, and injects the next micropore carrier by the same imbibition operation, and continuously operates until the number of the injected micropore carriers reaches the number required by the test;

sixthly, marking the qualified microporous carrier: taking the micro-porous plate after the injection from the clamping tool, placing a linear tool below the micro-porous plate, inverting the micro-porous plate, directing the bottom surface of the plate to an operator, observing the straight line through the injection micro-porous carrier, observing that the micro-porous carrier bent or bent linearly is an agar block with bubbles or without flattening, belonging to unqualified micro-porous carriers, marking the bottom of the micro-pore by using a marker pen, and marking the unmarked micro-porous carrier as a qualified micro-porous carrier;

2) loading the sample to be tested into the microporous carrier

Transferring the prepared drug sample liquid to be detected and the test control treatment sample liquid to an aseptic workbench, sucking 10 mu l of the sample liquid by using a pipette, placing the pipette in the middle of the surface of the qualified microporous carrier prepared in the step 1) under the condition that the pipette head does not touch the surface of the microporous carrier, automatically expanding and dispersing the pipette on the surface of the microporous carrier, and standing the pipette on the aseptic workbench to dry the drug sample liquid to form a drug-loaded microporous carrier;

3) loading pathogenic spores into microporous carrier

Under the aseptic condition, fully and uniformly suspending the prepared pathogenic bacteria spore liquid, sucking 1 mu l of the spore liquid by using a liquid transfer gun, and placing the liquid transfer gun in the middle of the medicine-carrying microporous carrier prepared in the step 2) under the condition that the gun head does not touch the surface of the microporous carrier to naturally disperse the spore liquid;

4) spore germination culture

Flatly placing the microporous plate after the operation of the step 3) in a clean moisturizing appliance, and transferring the microporous plate into an incubator for constant-temperature culture until the spores subjected to blank control treatment fully germinate;

5) observation of results

Taking out the culture material obtained in the step 4) from the incubator, opening a cover of a moisturizing device, inversely placing a microporous plate carrying the microporous carrier on an objective table of a microscope, aligning a lens of an objective lens to the bottom surface of the micropore, adjusting a focus of the lens to the spores on the surface of the microporous carrier, and observing and recording the germination condition of the spores on each microporous carrier; the inhibitory effect of the tested drugs on spore germination was calculated from the control-treated data.

Technical Field

The invention relates to a plant pathology technology, in particular to a determination method for a microporous carrier detection inhibitor.

Technical Field

Currently and for a considerable period of time in the future, pesticide control remains the main means for controlling plant diseases. Exploring and digging biogenic pesticides are important directions for pesticide control, and early exploration and digging of biogenic pesticides often collect or collect substances having inhibitory action on plant pathogenic bacteria from nature or separate active ingredients having inhibitory action from organisms or biological products. Obviously, the efficient and feasible inhibitor identification and detection means is beneficial to improving the exploration and excavation success and efficiency of the biological pesticide. The current common method for identifying the effect of the inhibitor on the pathogenic bacteria comprises a measuring method by utilizing an agar culture medium plate, and the main technical idea of the measuring method is to prepare a drug-containing culture plate by utilizing a common plate, then inoculate the pathogenic bacteria, and then culture and observe the inhibiting effect of the drug on the pathogenic bacteria. The technical scheme of the determination requires that the sample amount of a sample to be determined is large, the sample amount is too small, the sample cannot be detected, and the inhibitory active substances with small content are easy to miss or omit.

Disclosure of Invention

The invention aims to provide a determination method for detecting the inhibition effect of related substances on spore germination by using a microporous carrier technology.

The technical scheme for solving the technical problems is as follows:

a method for measuring the inhibitor of microporous carrier detection includes such steps as injecting 40 microliter agar culture medium into the micropores of a microporous plate to obtain microporous carrier, loading the medicinal sample and pathogenic spore on said microporous carrier, and measuring the inhibition of the medicinal sample on spore germination:

1. preparation of qualified microporous carrier by microporous plate

1) The micro-porous plate is clamped and stabilized: preparing a clamping and stabilizing tool which can clamp the microporous plate stably and flatly and has a clean surface, transferring the clamping and stabilizing tool to a sterile workbench, and clamping the clean microporous plate on the clamping and stabilizing tool.

2) Melting of agar medium: heating and melting agar culture medium filled in a triangular flask and suitable for spore germination of pathogenic bacteria.

3) Treatment of the medium at 80 ℃: heating a common water bath kettle in advance to be constant at 80 ℃, putting the culture medium melted in the operation 2) into the water bath kettle, and balancing to 80 ℃.

4) And (3) treatment of bubble-free pipetting by a pipetting gun: transferring the culture medium balanced to 80 ℃ in the operation 3) to a sterile workbench, adjusting the liquid taking amount of a liquid-transferring gun to 40 mu l, and extending a gun head into the culture medium at 80 ℃ to repeatedly suck and discharge the culture medium until no bubble phenomenon occurs in liquid discharging operation.

5) Preparation of microporous carrier: and 4) after no bubble phenomenon exists, excessively pressing the pipette gun to suck the culture medium, lifting the pipette head, removing the culture medium adhered to the outside of the pipette head by adhering the pipette head to the wall of the opening of the triangular flask, transferring the pipette head into the micropores of the microporous plate, injecting 40 microliter of culture medium to the bottom of the micropores, keeping the pipette head in the culture medium liquid to move circularly, guiding the culture medium to be uniformly distributed on the bottom surface of the micropores, and cooling to form a tiny flat plate of the agar culture medium as a micropore carrier for testing. After a micropore is injected, the mother finger presses the pipette gun, returns to and moves to the culture medium in the triangular flask at the temperature of 80 ℃, the next micropore carrier is injected by the same pipetting operation, and the operation is continued until the number of the injected micropore carriers reaches the number required by the test.

6) Identification of qualified microporous support: the injection-finished microporous plate of the operation 5) is taken out of the clamping and stabilizing tool, a linear tool is placed below the microporous plate, the microporous plate is reversed, the bottom surface of the plate faces an operator, the linear is observed through the injection-finished microporous carrier, the curved or bent microporous carrier is observed to be an agar block with bubbles or without flattening, the microporous carrier belongs to an unqualified microporous carrier, a marker pen is used for marking the bottom of the micropore, and the unlabeled microporous carrier is a qualified microporous carrier.

2. Loading the sample to be tested into the microporous carrier

Transferring the prepared drug sample liquid to be detected and the test control treatment sample liquid to an aseptic workbench, sucking 10 mu l of the sample liquid by using a pipette, placing the pipette in the middle of the surface of the qualified microporous carrier prepared in the step (1) under the condition that the pipette tip does not touch the surface of the microporous carrier, automatically expanding and dispersing the pipette on the surface of the microporous carrier, and standing the microporous carrier on the aseptic workbench to dry the drug sample liquid to form the drug-loaded microporous carrier.

3. Loading spores into a microporous carrier

And (3) under the aseptic condition, fully and uniformly suspending the prepared pathogenic bacteria spore liquid, sucking 1 mu l of the spore liquid by using a liquid transfer gun, and placing the liquid in the middle of the medicament-carrying microporous carrier prepared in the step (2) under the condition that the gun head does not touch the surface of the microporous carrier to naturally disperse the spore liquid.

4. Spore germination culture

And (4) flatly placing the microporous plate after the operation of the step (3) in a clean moisturizing appliance, and transferring the microporous plate into an incubator for constant-temperature culture until the spores treated by the blank control germinate sufficiently.

5. Observation of results

Taking out the culture material obtained in the step (4) from the incubator, opening a cover of the moisturizing device, inversely placing the micropore plate carrying the micropore carrier on an object stage of a microscope, aligning the lens of the objective lens to the bottom surface of the micropore, adjusting the focus of the lens to the spores on the surface of the micropore carrier, and observing and recording the germination condition of the spores on each micropore carrier; the inhibitory effect of the tested drugs on spore germination was calculated from the control-treated data.

The invention has the advantages that:

1) the test system is miniaturized: the basic test unit of the conventional flat plate measurement technology is a set of plates, more than 3 repeats are usually arranged, and more than 3 sets of plates are needed for one sample, so that more culture instruments, more consumables and more culture space are occupied; the basic test unit of the technology of the invention is a micropore carrier, 96 micropore carriers can be prepared by a conventional micropore plate, and the miniaturization of a test system can be realized.

2) And (3) detection sample micro-quantification: one test unit of the conventional plate measurement technology usually needs to consume more than 10000 mul of sample liquid, while one test unit of the technology only consumes 10 mul of sample liquid with little dosage.

Detailed description of the preferred embodiments

The present invention will be further described with reference to the following examples.

The invention uses the micro-hole of the conventional micro-hole plate to prepare the agar culture medium micro-plate with the volume of 40 mul, and the micro-plate is loaded with the drug sample and the pathogenic bacteria spore to carry out the inhibition test, thereby the micro-plate is called as the micro-hole carrier.

The micropore carrier used for testing needs to have consistent volume and size, the smaller the volume is, the better the volume is, the uniform distribution is on the bottom surface of the micropore, the surface is flat, and no air bubble is in the micropore. The inventor's original trial practice found that there are technical problems to be solved in order to prepare a qualified microporous carrier, such as how much culture medium should be added to a single micropore? How high temperature should be taken for the medium to be filled? How to check whether the microporous support is acceptable? The inventor tries the transfer gun transfer technology to prepare, and has some technical obstacles: firstly, conventional pipetting operation is carried out, and when agar culture medium in a hot-melt state is transferred, air bubbles are easily formed on the micropore carrier; secondly, the culture medium discharged by the gun head is easily adhered to one side of the micropore and is not flat; thirdly, when the temperature is low, the agar culture medium sucked in the gun head is easy to solidify and cannot be discharged, or the agar culture medium is solidified quickly and cannot be expanded sufficiently after being discharged; fourthly, when the temperature is higher, the operation is easier to form air bubbles in the discharged agar culture medium; fifthly, during the suction operation, more culture medium liquid is adhered to the outer wall of the gun head and is transferred and released along with the gun head, so that the accuracy of the liquid transfer amount is influenced. How to solve the technical problems is not disclosed with a related technical scheme so far, so that the preparation of qualified microporous carriers becomes the technical key of the invention.

Repeated research shows that the technical problems can be solved by adopting the following technical method: sucking 40 mul of agar culture medium at 80 ℃ by using a pipette gun, injecting the agar culture medium into the bottom of the micropore, and directly guiding the culture medium to be flat by using a gun head; and (5) checking whether the microporous carrier is qualified or not by using a straight line observation and comparison method. The specific operation method comprises the following steps:

prepare a instrument that can block steady micropore board, the purpose prevents and restricts the micropore board and removes at the preparation in-process because when the culture medium that the rifle head guide injected into the micropore bottom with the pipette is flat, whole micropore board can follow and remove. On the sterile working table, the microporous plate is stably placed in a clamping and stabilizing tool for the next operation; the agar medium contained in the flask was melted by heating and transferred to a prepared 80 ℃ water bath, and the medium was equilibrated to 80 ℃. Then transferring the culture medium with the temperature of 80 ℃ to a sterile workbench, taking out a proper pipetting gun, adjusting the pipetting quantity to 40 mu l, extending the gun head into the culture medium with the temperature of 80 ℃, slightly and repeatedly sucking/discharging, observing the bubble emergence condition of the liquid level of the culture medium, repeatedly sucking/discharging until no bubble phenomenon appears, normally sucking/discharging for about 20 times to reach balance, at the moment, slightly pressing down the pipetting quantity, slightly moving the gun head, slightly sticking the gun head on the wall of the mouth of the triangular flask and slightly staying for about 1 second, removing the culture medium adhered to the outside of the gun head, then transferring the culture medium into the micropores of the clamped and stable microporous plate, slightly pressing the gun to discharge 40 mu l of the culture medium into the bottom of the micropores, normally piling the injected culture medium on one side of the bottom, at the moment, keeping the pipetting gun in the pressed state, keeping the gun head in the culture medium, firstly rotating along the circular line at the periphery of the bottom of the micropores, and gradually rotate around the center to guide the culture medium to be uniformly distributed on the bottom surface of the micropore, and the purpose can be achieved by rotating around 3-5 circles. After cooling, the culture medium can form a tiny agar block with a flat surface and no air bubbles inside, and becomes a qualified micropore carrier suitable for testing. After one micropore carrier is injected, the mother finger presses the liquid transfer gun, returns to the culture medium in the triangular flask at the temperature of 80 ℃, sucks and transfers the liquid to inject the next micropore carrier, and continues the operation until the number of the injected micropore carriers reaches the number required by the test, the operation can not be stopped for a long time in the midway, otherwise the culture medium is solidified at the gun head, once the solidification occurs, the gun head needs to be replaced, the bubble removal operation is carried out again, and then the next micropore carrier is injected by liquid absorption. And after the injection is finished, taking the microporous plate out of the clamping and stabilizing tool for the next quality inspection operation.

Because the micropore plate and the agar culture medium are transparent, the micropore carrier is thin and deep at the bottom of the micropore, and whether the micropore carrier is qualified or not is difficult to observe and judge directly from the opening of the micropore. In continuous exploration and observation, when the normal straight line penetrates through the bubble or uneven micropore carrier to enter the eyes of an observer, the straight line has bending or bending phenomenon, a convenient method for inspecting the qualification of the microporous carrier is found at one time, the specific operation method is that the injection finished microporous plate is inverted, the bottom surface of the microporous plate faces to an operator and is vertical to the sight direction of the operator, a straight line tool (such as a straight edge line or a straight line drawn by a pen on white paper) is placed on one side of the front face of the micropore plate, an operator observes the straight line through the injected micropore carrier, observes the micropore carrier with the bent or bent straight line, is an agar block with air bubbles or an agar block without uniform flattening, belongs to an unqualified micropore carrier, marks the micropore at the bottom by a marker pen, and is a qualified micropore carrier after the inspection is finished and the carrier on the unmarked micropore is a qualified micropore carrier.

After preparing a qualified microporous carrier, loading a drug into the microporous carrier, and specifically, putting a prepared drug sample to be detected and a test control treatment sample into a workbench, sucking 10 mu l of sample liquid by using a pipette gun, gently placing the sample liquid in the middle of the surface of the microporous carrier, and taking care to avoid the gun head from touching the surface of the microporous carrier.

The drug-loaded microporous carrier can then be loaded with spores of pathogenic bacteria. The specific method is that the prepared pathogenic bacteria spore liquid is taken, the spores are shaken to be fully and uniformly suspended, 1 mul of spore liquid is sucked by a liquid transfer gun on a sterile workbench, and the spore liquid is lightly spotted in the middle of the drug-carrying micropore carrier like the prior liquid transfer spotting of the drug sample liquid, so that the spore liquid is naturally dispersed. Due to the small amount of spore liquid, the dispersion range does not usually exceed that of the drug sample. Practical work shows that the subsequent observation results are facilitated by dispersing about 100 spores per microporous carrier surface, so that the spore concentration of the prepared spore liquid is adjusted to 1 x 10⁶One per ml.

After the above steps are finished, the microporous plate is stably placed in a moisture-preserving apparatus prepared in advance, the apparatus is kept in a stable state, and the apparatus is transferred to an incubator for constant-temperature culture, so that spores are promoted to germinate. Because of different germination characteristics of the spores of different pathogenic bacteria, the culture time of different pathogenic bacteria is different, and the spores are cultured until the blank control treated spores are fully germinated in principle. Then observing results, placing the microporous plate under a microscope, aligning the lens with the bottom surface of the micropore, adjusting an object stage or the lens to enable the focal point of the lens to be aligned to the spores on the surface of the micropore carrier, and observing and recording the germination conditions of the spores on each micropore carrier; the inhibitory effect of the tested drugs on spore germination was calculated from the control-treated data.