A bioassay device and method for detecting the sensitivity of the American white moth to Cry1Ab toxin

Abstract

The present invention relates to a bioassay device and method for detecting the sensitivity of the gypsy moth to Cry1Ab toxin, and relates to the technical field of gypsy moth control, comprising a test box, and the interior of the test box is equally divided into four test cavities with openings facing upward; a capping assembly arranged on the test box and a reagent tube group clamped on the capping assembly; a driving assembly arranged directly above the reagent tube group, the driving assembly comprising a rotating shaft and a pushing piece arranged on the rotating shaft, and the pushing piece slides with the tube wall of each reagent tube of the reagent tube group under the push of the rotating shaft; the capping assembly comprises a swing frame, a capping piece and a locking seat, and a rotating piece is arranged in the locking seat, and the locking seat is rotated so that one end of the rotating shaft is connected to the rotating piece, and the locking seat drives the swing frame to rotate to control the opening of the capping piece. The present invention can realize the batch determination of the sensitivity of the gypsy moth to Cry1Ab toxin, and the test flexibility is strong and the efficiency is high.

Description

Technical Field

[0001] The invention belongs to the technical field of gypsy moth control, and in particular relates to a bioassay device and method for detecting the sensitivity of gypsy moth to Cry1Ab toxin. Background Art

[0002] The gypsy moth is a destructive pest and is listed as an international quarantine target. Currently, the development of some biological insecticides can effectively control the occurrence of gypsy moth disasters. For example, the δ-endotoxin produced by Bacillus thurigensis (Bt) has a high insecticidal activity against the gypsy moth, while being safe to natural enemies and humans and animals. Based on this idea, if transgenic Bt crops are developed, the damage caused by the gypsy moth can be better controlled.

[0003] With the large-scale planting of Bt transgenic crops, the gypsy moth has developed resistance to Bt protein. Therefore, it is necessary to detect the sensitivity of the gypsy moth to Cry1Ab toxin to provide a theoretical basis for the comprehensive management of the gypsy moth. To this end, the present invention provides a bioassay device and method for detecting the sensitivity of the gypsy moth to Cry1Ab toxin to achieve efficient and batch detection. Summary of the invention

[0004] The purpose of the present invention is to provide a bioassay device and method for detecting the sensitivity of the American white moth to Cry1Ab toxin in order to solve the above problems so as to achieve efficient and batch detection.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0006] The present invention provides a bioassay device for detecting the sensitivity of the American white moth to Cry1Ab toxin, comprising a test box, wherein the interior of the test box is equally divided into four test chambers with openings facing upwards;

[0007] A cover assembly provided on the test box and a reagent tube set clamped on the cover assembly, wherein the number of reagent tubes in the reagent tube set is consistent with the number of test cavities;

[0008] A driving assembly is arranged directly above the reagent tube group, the driving assembly comprises a rotating shaft and a pushing piece arranged on the rotating shaft, and the pushing piece is slidably matched with the tube wall of each reagent tube of the reagent tube group under the push of the rotating shaft;

[0009] The cover assembly includes a swing frame and a cover member. A locking seat is connected to the outer side of the swing frame, and a rotating member is arranged in the locking seat. When the locking seat is rotated so that one end of the rotating shaft is connected to the rotating member, the locking seat drives the swing frame to rotate to control the opening of the cover member.

[0010] As a further optimization scheme of the present invention, the structure of a single reagent tube in the reagent tube group includes an outer tube body, a tapered tube arranged at the lower end of the outer tube body, and a jet nozzle arranged at the lower end of the tapered tube.

[0011] As a further optimization scheme of the present invention, the pushing member includes a cross frame, a nut seat arranged on the cross frame, and a threaded sleeve which is sleeved on the rotating shaft and threadedly cooperates with the nut seat. The lower end surface of the cross frame is provided with a pushing rod corresponding to the number of reagent tubes in the reagent tube group, and the upper end of the rotating shaft is driven to rotate by connecting to a driving motor.

[0012] As a further optimization scheme of the present invention, the locking seat includes an axle connection seat, the end of the rotating shaft rod extending into the axle connection seat is provided with an external threaded portion, the inner side wall of the axle connection seat is provided with an internal threaded portion matching the external threaded portion, the rotating part includes a ball seat provided in the axle connection seat and a ball block movably provided in the ball seat, the lower end of the rotating shaft rod is provided with a plug-in rod plugged into the ball block, the outside of the rotating shaft rod is provided with a sleeve, and the distance of meshing transmission between the external threaded portion and the internal threaded portion of the rotating shaft rod is equal to the plug-in distance of the plug-in rod from the ball block.

[0013] As a further optimization scheme of the present invention, the sealing member includes a top cover, the top cover is provided with a through hole corresponding to the position of the test cavity, and four partition frames are evenly distributed on the top cover, a partition cover is rotatably fitted inside the partition frame, a swing rod connected to the partition cover is provided on the outside of the swing frame corresponding to the outside of the partition frame, and a partition net corresponding to the through hole is provided on the partition cover.

[0014] As a further optimization solution of the present invention, the partition frame includes a frame plate, the interior of the frame plate is rotatably matched with a rotating shaft by means of a torsion spring, and one end of the partition cover is connected to the rotating shaft and wound around the rotating shaft.

[0015] As a further optimization solution of the present invention, a manure accumulation plate is embedded at the lower end of the test box, and a foraging plate is provided in each test cavity of the test box.

[0016] The present invention also provides a method for detecting the sensitivity of the American white moth to CryAb toxin using any of the above-mentioned bioassay devices, comprising the following steps:

[0017] Step 1: Place the white moth larvae grouped in the same number into each test cavity of the test box and keep them in the same feeding environment;

[0018] Step 2: Prepare CryAb feeds of different concentrations, take the same amount and place them in each reagent tube of the reagent tube group, insert one end of the rotating shaft into the locking seat so that the pushing piece extends into the tube mouth of each reagent tube accordingly, rotate the locking seat so that one end of the rotating shaft is connected to the rotating piece, and the locking seat drives the swing frame to rotate to control the opening of the cover piece, so that each test cavity opening is in an open state;

[0019] Step 3: After the rotating shaft is rotated, the pushing member is driven to push the CryAb feed in each reagent tube into each test cavity opening for feeding by the white moth larvae in each test cavity. The rotating shaft stops rotating, and the locking seat is rotated so that one end of the rotating shaft is disconnected from the rotating member. At the same time, the locking seat drives the swing frame to rotate to control the closing of the cover member, so that each test cavity opening is in a closed state.

[0020] Step 4: Set an observation period, observe the survival rate of the gypsy moth larvae after feeding, and determine the sensitivity of the gypsy moth to Cry1Ab toxin.

[0021] The beneficial effects of the present invention are:

[0022] (1) The present invention drives the push rod by rotating the shaft to push the artificial reagent mixed with the test reagent in each reagent tube of the test tube group into each test cavity of the test box at the same time. The operation is simple and fast, and can be carried out in batches with high efficiency.

[0023] (2) The structure of the device of the present invention is highly flexible. When the locking seat is rotated so that one end of the rotating shaft is connected to the rotating member, the locking seat drives the swing frame to rotate to control the opening of the cover member, which does not hinder the pushing of the reagent into the test chamber and can ensure the sealing of the test chamber after the reagent is pushed.

[0024] (3) The bioassay method provided by the present invention can not only realize the batch determination of the sensitivity of the gypsy moth to Cry1Ab toxin at the same concentration or concentration, but also realize the batch determination of the sensitivity of the gypsy moth to Cry1Ab toxin at different concentrations or different dosages, with strong test flexibility and high efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram of the overall structure provided by the present invention;

[0026] Figure 2 A schematic diagram of the connection between the locking seat and the driving assembly provided by the present invention;

[0027] Figure 3 A schematic diagram of the structure of the cover assembly provided by the present invention;

[0028] Figure 4 A three-dimensional structural diagram of the test box provided by the present invention;

[0029] Figure 5 A schematic diagram of the internal structure of the partition frame provided by the present invention;

[0030] In the figure: 1. test box; 2. cross partition; 3. reagent tube group; 31. outer tube body; 32. conical tube; 33. jet nozzle; 4. drive assembly; 41. cross frame; 42. push rod; 43. rotating shaft; 44. nut seat; 45. drive motor; 46. screw sleeve; 47. shaft sleeve; 48. external threaded part; 49. plug-in rod; 5. capping assembly; 51. top cover; 52. swing frame; 53. swing rod; 53; 54. partition frame; 541. frame plate; 542. rotating shaft; 543. torsion spring; 55. partition cover; 56. partition net; 6. locking seat; 61. shaft seat; 62. ball seat; 63. ball block; 64. internal threaded part; 7. dung accumulation board; 8. foraging board. DETAILED DESCRIPTION

[0031] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0032] Example 1

[0033] like Figure 1-3 As shown, the present embodiment provides a bioassay device for detecting the sensitivity of the American white moth to Cry1Ab toxin, comprising a test box 1, wherein the interior of the test box 1 is divided into four test chambers with openings facing upward by a cross partition 2;

[0034] A cover assembly 5 is provided on the test box 1 and a reagent tube set 3 is clamped on the cover assembly 5, and the number of reagent tubes in the reagent tube set 3 is consistent with the number of test cavities;

[0035] A driving assembly 4 is arranged directly above the reagent tube group 3, and the driving assembly 4 includes a rotating shaft 43 and a pushing piece arranged on the rotating shaft 43, and the pushing piece is slidably matched with the tube wall of each reagent tube of the reagent tube group 3 under the push of the rotating shaft 43;

[0036] like Figure 4-5 As shown, the cover assembly 5 includes a swing frame 52 and a cover member. A locking seat 6 is connected to the outer side of the swing frame 52. A rotating member is provided inside the locking seat 6. When the locking seat 6 is rotated so that one end of the rotating shaft 43 is connected to the rotating member, the locking seat 6 drives the swing frame 52 to rotate to control the opening of the cover member.

[0037] Specifically, the structure of a single reagent tube in the reagent tube group 3 includes an outer tube body 31, a conical tube 32 arranged at the lower end of the outer tube body 31, and a jet nozzle 33 arranged at the lower end of the conical tube 32. The reagent tube group 3 composed of multiple reagent tubes can be used to determine the sensitivity of the gypsy moth to Cry1Ab toxin at different concentrations or different dosages. The setting of the jet nozzle 33 helps to quickly put the artificial feed mixed with the test reagent into the test cavity.

[0038] like Figure 1 As shown, the pushing member includes a cross frame 41, a nut seat 44 arranged on the cross frame 41, and a screw sleeve 46 which is sleeved on the rotating shaft 43 and threadedly matched with the nut seat 44. The lower end surface of the cross frame 41 is provided with a pushing rod 42 corresponding to the number of reagent tubes of the reagent tube group 3. The upper end of the rotating shaft 43 is driven to rotate by connecting to a driving motor 45. When in use, after the driving motor 45 drives the rotating shaft 43 to rotate, the screw sleeve 46 on its outside rotates therewith, so that the nut seat 44 on the screw sleeve 46 produces a vertical displacement. The nut seat 44 is installed on the cross frame 41, so that the cross frame 41 together with the pushing rod 42 on the cross frame 41 can also produce a vertical displacement, and the pushing rod 42 is in a state of abutting and sliding fit with the tube wall of the reagent tube, so that the test reagent in the reagent tube can be pushed out when the pushing rod 42 produces a vertical displacement.

[0039] like Figure 2 As shown, the locking seat 6 includes an axle connection seat 61, one end of the rotating shaft rod 43 extending into the axle connection seat 61 is provided with an external threaded portion 48, the inner side wall of the axle connection seat 61 is provided with an internal threaded portion 64 matched with the external threaded portion 48, the rotating member includes a ball seat 62 arranged in the axle connection seat 61 and a ball block 63 movably arranged in the ball seat 62, the lower end of the rotating shaft rod 43 is provided with a plug-in rod 49 plugged with the ball block 63, the outside of the rotating shaft rod 43 is provided with a shaft sleeve 47, and the meshing transmission distance of the external threaded portion 48 of the rotating shaft rod 43 and the internal threaded portion 64 is equal to the plug-in distance of the plug-in rod 49 from the ball block 63;

[0040] The cover member includes a top cover 51, on which a through hole corresponding to the position of the test cavity is opened, and four partition frames 54 are evenly distributed on the top cover 51, and a partition cover 55 is rotatably matched inside the partition frame 54, and a swing rod 53 connected to the partition cover 55 is provided on the outer side of the swing frame 52 corresponding to the outer side of the partition frame 54, and a partition net 56 corresponding to the through hole is provided on the partition cover 55;

[0041] When in use, the shaft seat 61 is rotated, on the one hand, the swing frame 52 outside thereof is driven to rotate, so that the swing rod 53 outside the swing frame 52 sends the partition cover 55 into the partition frame 54, so that the opening corresponding to each test cavity on the top cover 51 is opened. On the other hand, when the shaft seat 61 is rotated, the internal threaded portion 64 inside thereof is meshed with the external threaded portion 48 at the lower end of the rotating shaft rod 43, so that the rotating shaft rod 43 is displaced and extends into the shaft seat 61. When the internal threaded portion 64 and the external threaded portion 48 are meshed and transmitted, the rotating shaft rod 43 is displaced and extends into the shaft seat 61. The plug-in rod 49 at the lower end of 43 is just plugged into the ball block 63, and the ball block 63 is provided with a hole for the plug-in rod 49. At the same time, the shaft sleeve 47 arranged outside the rotating shaft rod 43 is movably plugged into the upper end opening of the shaft connection seat 61. When the rotating shaft rod 43 is driven to rotate, the rotating shaft rod 43 together with the ball block 63 plugged into the lower end thereof and the ball seat 62 rotate and cooperate, which does not affect the transmission structure of the shaft connection seat 61 and the sealing member, and can also ensure that the rotating shaft rod 43 rotates to drive the pushing member to push the test reagent in the reagent tube into each test cavity;

[0042] After the test reagent is pushed, the rotation of the rotating shaft 43 is stopped, and the rotating shaft 43 is lifted up to disengage the plug-in rod 49 from the ball block 63. At the same time, the external threaded portion 48 of the rotating shaft 43 contacts the internal threaded portion 64. At this time, the rotating shaft seat 61 causes the swing rod 53 of the swing frame 52 on its outer side to pull the partition cover 55 out of the partition frame 54, leaving only the partition net 56 on the partition cover 55 for ventilation of the test chamber. At the same time, the external threaded portion 48 and the internal threaded portion 64 produce meshing transmission, and the rotating shaft 43 and the pushing piece can be respectively disengaged from the structural connection with the locking seat 6 and the reagent tube group 3. Subsequently, the reagent tube group 3 can be disassembled for subsequent cleaning.

[0043] like Figure 5 As shown, the partition frame 54 includes a frame plate 541, and the interior of the frame plate 541 is rotatably matched with a rotating shaft 542 by setting a torsion spring 543. One end of the partition cover 55 is connected to the rotating shaft 542 and is wound on the rotating shaft 542. By setting the torsion spring 543, the automatic reset property of the torsion spring 543 allows the partition cover 55 to be more smoothly retracted into the frame plate 541, and will not be stuck outside the partition frame 54 to affect the pushing of the test reagent.

[0044] like Figure 3 As shown, a feces accumulation plate 7 is embedded at the lower end of the test box 1 for receiving feces discharged by the gypsy moth larvae, and a feeding plate 8 is provided in each test cavity of the test box 1 for receiving artificial feed mixed with test reagents for the gypsy moth larvae to eat.

[0045] Example 2

[0046] This embodiment provides a method for detecting the sensitivity of the American white moth to Cry1Ab toxin using any of the above-mentioned bioassay devices, comprising the following steps:

[0047] Step 1: Place the white moth larvae grouped in the same number into each test cavity of the test box 1 and keep them in the same feeding environment;

[0048] Step 2: Prepare artificial feeds containing different concentrations of Cry1Ab, take the same amount and put it in each reagent tube of the reagent tube group 3, insert one end of the rotating shaft 43 into the locking seat 6 so that the pushing piece extends into the tube mouth of each reagent tube accordingly, rotate the locking seat 6 so that one end of the rotating shaft 43 is connected to the rotating piece, and at the same time, the locking seat 6 drives the swing frame 52 to rotate to control the opening of the cover piece, so that each test cavity opening is in an open state;

[0049] Step 3: After the rotating shaft 43 is rotated, the pushing member is driven to push the Cry1Ab feed in each reagent tube into each test cavity opening for the white moth larvae in each test cavity to eat. The rotating shaft 43 stops rotating, and the locking seat 6 is rotated so that one end of the rotating shaft 43 is disconnected from the rotating member. At the same time, the locking seat 6 drives the swing frame 52 to rotate to control the closing of the cover member, so that each test cavity opening is in a closed state.

[0050] Step 4: Set an observation period, raise the larvae at a temperature of 27±2°C, a relative humidity of 60%-70%, and a photoperiod of 14L:10D, observe the survival rate of the gypsy moth larvae after feeding, and determine the sensitivity of the gypsy moth to the Cry1Ab toxin.

[0051] The components and their weight percentages of the artificial feed are as follows: 5-12% germ, 2-5% sucrose, 3-7% protein, 0.3-1% Wechsler salt, 0.4-1.2% sorbic acid, 0.1-0.5% methyl paraben, 0.02-0.19% ascorbic acid, 0.03-0.15% vitamin B, 1.1-1.8% agar, 0.05-0.16% cholesterol, and the balance is water; Cry1Ab is an MVPII aqueous solution containing 20% ​​Cry1Ab type Bt ICP.

[0052] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A bioassay device for detecting the sensitivity of the nymphalid moth to Cry1Ab toxin, characterized in that: The test box (1) comprises a test box (1), wherein the interior of the test box (1) is equally divided into four test chambers with openings facing upwards by arranging a cross partition (2); A cover assembly (5) arranged on the test box (1) and a reagent tube group (3) clamped on the cover assembly (5), wherein the number of reagent tubes in the reagent tube group (3) is consistent with the number of test chambers; A driving assembly (4) is arranged directly above the reagent tube group (3), the driving assembly (4) comprising a rotating shaft (43) and a pushing piece arranged on the rotating shaft (43), the pushing piece being pushed by the rotating shaft (43) to abut against and slide against the tube wall of each reagent tube of the reagent tube group (3); The cover assembly (5) comprises a swing frame (52) and a cover member. The outer side of the swing frame (52) is connected to a locking seat (6). A rotating member is arranged inside the locking seat (6). When the locking seat (6) is rotated so that one end of the rotating shaft (43) is connected to the rotating member, the locking seat (6) drives the swing frame (52) to rotate to control the opening of the cover member.

2. A bioassay device for detecting the sensitivity of the nymphalid moth to Cry1Ab toxin according to claim 1, characterized in that: The structure of a single reagent tube in the reagent tube group (3) comprises an outer tube body (31), a tapered tube (32) arranged at the lower end of the outer tube body (31), and a jet nozzle (33) arranged at the lower end of the tapered tube (32).

3. A bioassay device for detecting the sensitivity of the nymphalid moth to Cry1Ab toxin according to claim 1, characterized in that: The pusher comprises a cross frame (41), a nut seat (44) arranged on the cross frame (41), and a screw sleeve (46) sleeved on a rotating shaft (43) and threadedly matched with the nut seat (44); a pusher rod (42) corresponding to the number of reagent tubes in the reagent tube group (3) is arranged on the lower end surface of the cross frame (41); and the upper end of the rotating shaft (43) is driven to rotate by connecting to a driving motor (45).

4. A bioassay device for detecting the sensitivity of the nymphalid moth to Cry1Ab toxin according to claim 3, characterized in that: The locking seat (6) includes an axle connection seat (61), one end of the rotating shaft (43) extending into the axle connection seat (61) is provided with an external threaded portion (48), the inner side wall of the axle connection seat (61) is provided with an internal threaded portion (64) matching the external threaded portion (48), the rotating member includes a ball seat (62) arranged in the axle connection seat (61) and a ball block (63) movably arranged in the ball seat (62), the lower end of the rotating shaft (43) is provided with a plug-in rod (49) plugged into the ball block (63), the outside of the rotating shaft (43) is provided with a sleeve (47), and the meshing transmission distance of the external threaded portion (48) and the internal threaded portion (64) of the rotating shaft (43) is equal to the plug-in distance of the plug-in rod (49) from the ball block (63).

5. A bioassay device for detecting the sensitivity of the nymphalid moth to Cry1Ab toxin according to claim 1, characterized in that: The sealing member comprises a top cover (51), the top cover (51) is provided with a through hole corresponding to the position of the test cavity, and four partition frames (54) are evenly distributed on the top cover (51), a partition cover (55) is rotatably matched inside the partition frame (54), a swing rod (53) connected to the partition cover (55) is provided on the outer side of the swing frame (52) corresponding to the outer side of the partition frame (54), and a partition net (56) corresponding to the through hole is provided on the partition cover (55).

6. A bioassay device for detecting the sensitivity of the nymphalid moth to Cry1Ab toxin according to claim 5, characterized in that: The partition frame (54) comprises a frame plate (541), the interior of the frame plate (541) is rotatably matched with a rotating shaft (542) by means of a torsion spring (543), and one end of the partition cover (55) is connected to the rotating shaft (542) and is wound around the rotating shaft (542).

7. A bioassay device for detecting the sensitivity of the nymphalid moth to Cry1Ab toxin according to claim 1, characterized in that: A dung accumulation plate (7) is embedded at the lower end of the test box (1), and a foraging plate (8) is provided in each test cavity of the test box (1).

8. A method for detecting the sensitivity of the nymphalid moth to Cry1Ab toxin using the bioassay device according to any one of claims 1 to 7, characterized in that: The following steps are included: Step 1: Place the white moth larvae grouped in the same number into each test cavity of the test box and keep them in the same feeding environment; Step 2: prepare artificial feed containing different concentrations of Cry1Ab, take the same amount and place it in each reagent tube of the reagent tube group (3), extend one end of the rotating shaft (43) into the locking seat (6) so that the pushing piece extends into the tube mouth of each reagent tube accordingly, rotate the locking seat (6) so that one end of the rotating shaft (43) is connected to the rotating piece, and the locking seat (6) drives the swing frame (52) to rotate to control the opening of the cover piece, so that each test cavity opening is in an open state; Step 3: After the rotating shaft (43) is rotated, the pushing member is driven to push the Cry1Ab feed in each reagent tube into each test cavity opening for feeding by the white moth larvae in each test cavity. The rotating shaft (43) stops rotating, and the locking seat (6) is rotated so that one end of the rotating shaft (43) is disconnected from the rotating member. At the same time, the locking seat (6) drives the swing frame (52) to rotate to control the closing of the cover member, so that each test cavity opening is in a closed state. Step 4: Set an observation period, observe the survival rate of the gypsy moth larvae after feeding, and determine the sensitivity of the gypsy moth to Cry1Ab toxin.

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