CN113702626B - Simple and efficient biological assay reagent and method - Google Patents
Simple and efficient biological assay reagent and method Download PDFInfo
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- CN113702626B CN113702626B CN202010431147.1A CN202010431147A CN113702626B CN 113702626 B CN113702626 B CN 113702626B CN 202010431147 A CN202010431147 A CN 202010431147A CN 113702626 B CN113702626 B CN 113702626B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
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Abstract
The invention relates to the field of bioassays, in particular to a simple and efficient bioassay reagent and a method. The biological assay reagent is a liquid containing pesticide to be detected and edible pigment. When biological tests are carried out, the biological assay reagent is fed to the animals to be tested, the eating condition is observed, and when 85-95% of stomach toxicity of the animals to be tested is finished, normal foodstuff is fed to the animals to be tested, and relevant indexes are observed and counted. After the post-biological assay reagent is used, the stomach toxicity is easy and convenient to operate, the efficiency is high, and the stomach toxicity efficiency is improved by about 2 times. And the stomach toxicity amount is controllable, the stomach toxicity time is short, the difference of the stomach toxicity time among the individuals of the animal to be detected is small, and the time of ending the stomach toxicity can be estimated almost accurately. The advantages are beneficial to promoting the related research of biological pesticides and chemical pesticides and reducing the economic loss caused by the harm of farmland pests.
Description
Technical Field
The invention relates to the field of bioassays, in particular to a simple and efficient bioassay reagent and a method.
Background
Farmland pests are an important factor affecting agricultural production, and economic losses caused by the damage of farmland pests are serious every year. The pest comprehensive control is an important strategy for agricultural production, and plays an important role in sustainable development of agriculture and reduction of agricultural economic loss. The use of biopesticides, chemical pesticides, etc. for pest control has been an important research content in comprehensive pest control research, and at the same time, related research of biopesticides, chemical pesticides generally requires related bioassays.
In the traditional bioassay experiment, usually, research reagents are dripped on artificial feed for researching animals to be tested by means of the artificial feed for researching the animals to be tested, and bioassay is carried out.
However, at present, many researches on animals to be tested do not have proper artificial feed, and the traditional bioassay method has the defects of complicated operation, long toxin receiving experiment time, uncertainty in the toxin receiving ending time of the animals to be tested and the like, so that a certain limitation effect is generated on the researches based on the related bioassays. Therefore, the invention of a simpler and more efficient bioassay method is an urgent need for the current bioassay-based research.
The bioassay method has the defects of complicated operation, high requirements on the size, thickness and the like of the artificial feed, uncontrollable toxin receiving quantity, incomplete toxin receiving quantity, long toxin receiving time, large difference of toxin receiving time among the individuals of the animal to be detected, incapability of accurately estimating the time of ending the toxin receiving and the like.
Disclosure of Invention
First, the technical problem to be solved
In order to solve the problems in the existing bioassay method, the invention aims to provide a simple and efficient bioassay reagent and method, which are suitable for researching an animal to be tested without proper artificial feed to carry out a stomach toxicity experiment, and have the advantages of simple operation, short toxicity receiving time and relatively accurate record of the specific time of ending the toxicity receiving.
(II) technical scheme
The invention firstly provides a biological assay reagent which is liquid containing pesticide to be tested and edible pigment.
Preferably, when 1.8-2.2 mu L of edible pigment is added into 1mL of the bioassay reagent, the bioassay reagent not only has good indication function, but also has no significant influence on the growth and development of the animal to be tested, and is beneficial to improving the objectivity and accuracy of the measurement result.
Further preferably, 2.0. Mu.L of edible pigment is added per 1mL of the bioassay reagent.
Preferably, the color of the edible pigment is the same as or similar to the color of natural foodstuff of the animal to be tested, and the edible pigment can be red, blue or green.
Preferably, when the natural foodstuff of the animal to be tested is green, the edible pigment is preferably lemon yellow as a main component, and the concentration of the lemon yellow is 0.03 to 0.05mol/L (preferably 0.04 mol/L). The concentration has bright color and good indication effect, and is also favorable for attracting animals to be tested to take food.
The pesticide to be tested in the invention can be biological pesticide and/or chemical pesticide, the dosage of which can be determined according to the measurement requirement, and the method is not further limited.
The invention further provides a simple and efficient biological test method, wherein the biological test reagent is fed to the to-be-tested animals, the eating condition is observed, and when 85-95% of the to-be-tested animals are at the end of the inoculation, normal foodstuff is fed to the to-be-tested animals, and the relevant indexes are observed and counted.
Preferably, the animal to be tested is kept in a transparent keeping device.
Preferably, the test animal is starved prior to feeding the test animal with the bioassay reagent.
Preferably, the test animals not having consumed the bioassay reagent or the normal foodstuff are removed before the relevant index is observed and counted.
Some animals to be tested have a suitable artificial diet, and many animals to be tested do not have a suitable artificial diet. The normal foodstuff in the invention can be feed and/or natural foodstuff, and the natural foodstuff refers to foodstuff eaten by the animal to be tested in natural environment.
The invention further provides the use of the bioassay reagent or bioassay method in the bioassay of an animal, preferably an arthropod, more preferably the arthropod is a test animal.
The invention further provides application of the bioassay reagent or the bioassay method in farmland pest control.
(III) beneficial effects
After the use of the post-bioassay reagents of the present invention, the following advantages exist:
(1) The virus inoculation operation is simple and convenient, the efficiency is high, and the virus inoculation efficiency is improved by about 2 times.
(2) The inoculation amount is controllable, and whether the animal to be tested feeds the virus suspension completely can be clearly observed due to the indication effect of the edible pigment. If the feeding is not complete, marks are left in the culture device, so that the animal to be tested which is not completely inoculated with the toxin can be removed.
(3) The toxin receiving time is short, the indirect toxin time difference between the animal individuals to be tested is small, and the animal individuals to be tested can be ignored. And meanwhile, the time for ending the inoculation can be estimated almost accurately.
The advantages are beneficial to promoting the related research of biological pesticides and chemical pesticides and reducing the economic loss caused by the harm of farmland pests.
Drawings
FIG. 1 is a graph showing the comparison of the cumulative pupation rate of the bollworm larvae in example 1;
FIG. 2 is a comparison of the pupae weight of cotton bollworms in example 1;
FIG. 3 is a comparison of the cumulative mortality rate of the bollworm larvae in example 2.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
In the following embodiments, experiments and related invention description are performed using cotton bollworms, which are important cotton field pests, and specific insect virus, cotton bollworm nuclear polyhedrosis virus as examples.
Cotton bollworms (Helicoverpa armigera), which are lepidoptera, noctuidae, cotton bollworms insects, are widely distributed around the world and are world economic pests, which mainly endanger cotton, corn, vegetables, peanuts, and other crops, causing significant economic losses worldwide.
The cotton bollworm nuclear polyhedrosis virus (h. Armigera single nucleopolyhedrovirus, haSNPV) is a broad class of invertebrate DNA viruses. Has the advantages of strong insecticidal specificity, good insecticidal effect, vertical transmission, long lasting time, no harm to human and livestock and environment, and the like, and is widely studied. HaSNPV can infect hosts through a variety of pathways. Oral infection by feeding on foods contaminated with viruses is a major route of insect infection in nature. In particular, for viruses embedded in polyhedra, the viruses must pass through the insect digestive system and digest polyhedrin under the action of alkaline gastric juice, so that the viruses can be released and activated. According to this principle, oral infection is also the main mode of infection of hosts by the currently marketed biological agents of the hannpv virus.
The edible colorant used in the following embodiments is a schkman edible complex colorant purchased from foodborne, inc.
Throughout the bioassay process, the cotton bollworm growth environment remained consistent. The growth temperature is 26+/-1 ℃, the humidity is 40% -70%, and the photoperiod L/D=14/10.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
Example 1 no significant effect of edible pigment on the growth and development of Heliothis armigera
In this example, a treatment group and a control group were set up for bioassay study of the edible pigment.
Picking a sufficient number of cotton bollworms at the end of 3 years in advance in a plastic insect-raising box, and carrying out 12h starvation treatment for later use.
Treatment group: 1000 mu L ddH 2 O+2. Mu.L of the edible pigment was formulated as a solution.
The artificial feed was cut to 0.5cm by 0.1cm and 2.5 μl of the solution was added dropwise to the block. The feed is added into a plastic insect-raising box and fed to cotton bollworms (4 years old and 1 day) which are starved in advance.
Control group: 1000 mu L ddH 2 O.
Cutting artificial feed into 0.5cm 0.1cm size, and adding 2.5 μl ddH dropwise to the block 2 O. The feed is added into a plastic insect-raising box and fed to cotton bollworms (4 years old and 1 day) which are starved in advance.
Observing the eating condition (feeding time is controlled within 12 h), removing cotton bollworms which do not eat the processed feed, adding normal artificial feed into a bollworm raising box which eat the processed feed, putting the bollworms into a artificial climate box for raising and observing, and counting development indexes with the counting frequency of 1 time/24 h.
The treatment group and the control group each treated 15 heads, each repeated 3 times. The statistical results are shown in FIGS. 1-2.
As can be seen from FIG. 1 and the single factor variability results of spss, the treatment group fed edible pigment was fed with ddH 2 There was no significant difference in the cumulative pupation rate at each time point for the control group of O. The edible pigment has no significant influence on the pupation duration of the bollworm larvae.
As can be seen from FIG. 2 and the results of the spss one-factor differential analysis, the treatment group fed with the edible pigment was fed with ddH 2 There was no significant difference in pupal weight between the control group of O.
As can be seen from fig. 1 and 2, the edible pigment has no significant effect on the growth and development of cotton bollworms.
Example 2A simple and efficient bioassay using the method of the present invention
In this example, the conventional bioassay method and the bioassay method of the present invention were used to compare the two methods for the cotton bollworm stomach toxicity NaSNPV.
1. Reagent and material preparation
The concentration of the NasNPV suspension fed in this example was 4 x 10 7 The specific preparation method of the OBS/mL virus suspension comprises the following steps: naSNPV stock solution purchased from Keyun biosystems, inc. and having a concentration of 50 billion OBS/mL was used as the stock solution, and ddH was used 2 O was diluted in a gradient to give a concentration of 1000. Mu.L of 4X 10 7 OBS/mL virus suspension.
The specific preparation method of the bioassay reagent comprises the following steps: to the virus suspension was added 2. Mu.L of an edible pigment.
Picking a sufficient number of cotton bollworms at the end of 3 years in advance in a plastic insect-raising box, and carrying out 12h starvation treatment for later use.
2. Bioassay method and results
(1) Traditional bioassay methods: 2.5 mu L of virus suspension is dripped on artificial feed (0.5 cm 0.1 cm) cut in advance, cotton bollworms are fed for about 12 hours, cotton bollworms with stomach toxicity are picked up, normal artificial feed is added for feeding, and relevant growth indexes are observed and recorded.
(2) The bioassay method of the present invention: directly adding 2.5 mu L of bioassay reagent to the bottom of a plastic box containing prestarved cotton bollworms (4 years old and 1 day), and directly feeding.
Observing the eating condition, when most (90%) of cotton bollworms are at the end of the inoculation, adding normal artificial feed into the insect-breeding box with cotton bollworms, and removing cotton bollworms which do not eat the bioassay reagent or the treatment feed. And (5) putting the treated cotton bollworms into a climatic chamber for feeding and observing, and counting the development indexes with the counting frequency of 1 time/24 hours.
The conventional bioassay method and the bioassay method of the present invention were repeated 3 times each, and the statistical results are shown in fig. 3.
As can be seen from fig. 3 and the results of the spss single factor differential analysis, there is no significant difference in the cumulative mortality of each time period when bioassays are performed using the conventional method and the method of the present invention.
In addition, through the feeding process, the edible pigment is utilized to carry out the stomach toxicity experiment, and the average time for the end of the inoculation of each healthy cotton bollworm is within 10 minutes. In the traditional method, the artificial feed is used for carrying out the stomach toxicity experiment, and the average time for the end of the inoculation of each healthy cotton bollworm is about 12 hours. Therefore, the edible pigment is used for performing the stomach toxicity test, the difference of the individual time of the toxic end is small (within 10 min), while the traditional method is used for performing the stomach toxicity test, the difference of the individual time of the toxic end is large, and the accurate time of the toxic end is difficult to estimate.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (7)
1. A simple and efficient biological test method is characterized in that starvation treatment is firstly carried out on an animal to be tested, then the animal to be tested is directly fed with a biological assay reagent, the eating condition is observed, when 85-95% of the animal to be tested is at the end of poison receiving, the animal to be tested is fed with normal foodstuff, the animal to be tested which does not eat the biological assay reagent or the normal foodstuff is removed, and related indexes are observed and counted; the time for ending the inoculation is within 10 min; the animal to be tested is an insect;
the biological assay reagent is a liquid composed of pesticide to be detected and edible pigment;
the pesticide to be detected is a virus suspension;
the color of the edible pigment is the same as or similar to the color of the natural foodstuff of the animal to be tested;
1.8-2.2 mu L of edible pigment is added into 1mL of the bioassay reagent.
2. The bioassay method according to claim 1, wherein 2.0 μl of edible pigment is added per 1mL of said bioassay reagent.
3. The biological test method according to claim 1, wherein when the natural foodstuff of the animal to be tested is green, the edible pigment has a main component of lemon yellow, and the concentration of lemon yellow is 0.03-0.05 mol/L.
4. The biological test method of claim 1, wherein the test animal is housed in a transparent housing device.
5. The biological test method according to claim 1, wherein the normal foodstuff is a feed and/or a natural foodstuff.
6. Use of the biological test method according to any one of claims 1 to 5 in biological tests of insects.
7. Use of the biological test method according to any one of claims 1 to 5 for pest control in farmlands.
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| US6150126A (en) * | 1996-12-06 | 2000-11-21 | Wisconsin Alumni Research Foundation | Daphnia reproductive bioassay for testing toxicity of aqueous samples and presence of an endocrine disrupter |
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