AU2002318877B2 - Method and test kit for the detection of insecticide resistance in insect vectors - Google Patents
Method and test kit for the detection of insecticide resistance in insect vectors Download PDFInfo
- Publication number
- AU2002318877B2 AU2002318877B2 AU2002318877A AU2002318877A AU2002318877B2 AU 2002318877 B2 AU2002318877 B2 AU 2002318877B2 AU 2002318877 A AU2002318877 A AU 2002318877A AU 2002318877 A AU2002318877 A AU 2002318877A AU 2002318877 B2 AU2002318877 B2 AU 2002318877B2
- Authority
- AU
- Australia
- Prior art keywords
- buffer
- test kit
- propoxur
- acetylcholine
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
- 238000012360 testing method Methods 0.000 title claims description 27
- 241000238631 Hexapoda Species 0.000 title claims description 18
- 238000000034 method Methods 0.000 title claims description 16
- 239000013598 vector Substances 0.000 title description 7
- 238000001514 detection method Methods 0.000 title description 5
- 230000012865 response to insecticide Effects 0.000 title description 2
- 239000000872 buffer Substances 0.000 claims description 20
- ISRUGXGCCGIOQO-UHFFFAOYSA-N Rhoden Chemical compound CNC(=O)OC1=CC=CC=C1OC(C)C ISRUGXGCCGIOQO-UHFFFAOYSA-N 0.000 claims description 16
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 claims description 11
- 229960004373 acetylcholine Drugs 0.000 claims description 11
- SMBBQHHYSLHDHF-UHFFFAOYSA-M 2-acetyloxyethyl(trimethyl)azanium;iodide Chemical compound [I-].CC(=O)OCC[N+](C)(C)C SMBBQHHYSLHDHF-UHFFFAOYSA-M 0.000 claims description 10
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 claims description 9
- 108090000371 Esterases Proteins 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical group [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 4
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 3
- 235000011009 potassium phosphates Nutrition 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims 3
- 230000035945 sensitivity Effects 0.000 claims 2
- SLAMLWHELXOEJZ-UHFFFAOYSA-N 2-nitrobenzoic acid Chemical compound OC(=O)C1=CC=CC=C1[N+]([O-])=O SLAMLWHELXOEJZ-UHFFFAOYSA-N 0.000 claims 1
- GFZXQBDELXEPTQ-UHFFFAOYSA-N 3-[(3-carboxy-2-nitrophenyl)disulfanyl]-2-nitrobenzoic acid Chemical compound OC(=O)C1=CC=CC(SSC=2C(=C(C(O)=O)C=CC=2)[N+]([O-])=O)=C1[N+]([O-])=O GFZXQBDELXEPTQ-UHFFFAOYSA-N 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 239000000243 solution Substances 0.000 description 16
- 239000002917 insecticide Substances 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical group CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000003149 assay kit Methods 0.000 description 3
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 3
- 239000008057 potassium phosphate buffer Substances 0.000 description 3
- 241000255925 Diptera Species 0.000 description 2
- 208000011312 Vector Borne disease Diseases 0.000 description 2
- 150000004657 carbamic acid derivatives Chemical class 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 108010051152 Carboxylesterase Proteins 0.000 description 1
- 102000013392 Carboxylesterase Human genes 0.000 description 1
- 208000001490 Dengue Diseases 0.000 description 1
- 206010012310 Dengue fever Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 201000006353 Filariasis Diseases 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 208000025729 dengue disease Diseases 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- -1 organo phosphates Chemical class 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
OurRef: IRN 7758390 P/00/011 Regulation 3:2
AUSTRALIA
Patents Act 1990
ORIGINAL
COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Address for Service: Invention Title: Institute for Medical Research Jalan Pahang, 50588 Kuala Lumpur Wilayah Persekutuan Malaysia DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Method and test kit for the detection of insecticide resistance in insect vectors The following statement is a full description of this invention, including the best method of performing it known to me:- METHOD AND TEST KIT FOR THE DETECTION OF INSICTICIDE RESISTANCE IN INSECT VECTORS This invention relates to a method and test kit for determining intsect resistance to insecticides and more particularly provides a method and test kit for determining insect resistance to organophosphate and carbamate based inse cticides.
BACKGROUND TO THE INVENTION Chemical insecticides remain as the most important agents for the control of 0 vector-borne diseases such as malaria, dengue, filariasis etc. Countless deaths from these diseases have been spared since the introduction o the first synthetic insecticide. DDT in the 1940's. Prolonged use of insecticides has however, induced the development of resistance in the vectors, primarily nsects, that carry such diseases, which renders them less effective in combatir the vectors and thus the diseases that they spread. Resistance detection is mainly based on the World Health Organisation (WHO) standard test procedures w hich, among other things, are not user-friendly, are time-consuming; requ re many insect specimens, use test kits of short shelf life and need skilled manpower to conduct the tests and interpret the test results. As a result, very often I tle is known of the susceptibility status of many insect vectors, which tends to aff(t the outcomes of J control programmes.
The development of rapid field test kits would be a major breakthrough in the control of vector-borne diseases.
In general, the biochemical basis of resistance is due to one of three mechanisms; 1) increased level of non-specific esterases targeted against organophosphates and carbamates; 2) elevated activity of mixed-function oxidases against pyrettoids; and 3) non-susceptibility of acetylcholine esterase against organo phosphates and carbamates.
SUMMARY OF THE INVENTION This invention provides a method and test kit for determinincl susceptibility of acetylcholine esterase in insects using a single insect, that is (luick and easy to use and produces results that are very simple to interpret.
According to the invention the test kit comprises a cdntainer containing separate containers of: 1. a neutral to acid pH phosphate buffer solution II. acetylcholine iodide II. 5,5-dithio-bis-(2-nitrobenzoic acid) IV. propoxur (a carbamate based insecticide) and V. a water-miscible organic solvent.
For field use the kit will also contain the equipment for carrying out the test, for example mixing containers, test tubes, for example Eppend:'rf tubes, pipettes and a micro-assay plate.
The buffered pH is preferably between 6 and 7, more preferab y about 6.8.
The pH buffer is preferably potassium phosphate The propoxur is preferably used as solution of 5 to 25 mg/I in ethanol.
The water-miscible organic solvent is preferably acetone.
The method of the invention comprises the steps, in any order, :f homogenizing a single insect in the buffer, preferably by grinding it in a small ube, such as an Eppendorf tube, with a pestle, producing a solution of acetyl:holine iodide in buffered solvent, producing a solution of 5,5-dithio-bis-(2-nitrobenzoic acid) in the buffer, and producing a solution of propoxur in the buffer, and t-len dropping the insect homogenisate into the wells of an assay plate, dropping Ihe acetylcholine iodide and 5,5-dithio-bis-(2-nitrobenzoic acid) solutions into s:ime of the wells and 5,5-dithio-bis-(2-nitrobenzoic acid), acetylcholine iodid,. and propoxur solutions into the other wells and checking for a differeni;e in the yellow coloration between the samples in the two sets of cells.
_J
If desired, the propoxur and acetylcholine iodide solutions may ble pre-mixed.
The results may be determined by eye or, if more accurate re".ults are needed, by scanning using an immunoassay reader at 410 nm.
If the yellow coloration in the cells containing propoxur becorr ,s lighter after an incubation period the enzyme acetylcholine esterese is susceptible and the insect is not resistant to organophosphate and carbamate based insecticides.
The invention has the advantage of being the first availab'e field test kit for testing for acetylcholine esterase susceptibility that requires oily a single insect for testing and that can be used In a test that gives rapid test results (they are usually obtainable within 30 minutes) that can be r-lad visually and colorimetricatlywithout equipment. The test kits and method also require less skilled manpower and are easy to use without special training. Moreover, the kits have a long shelf life and are stable for weeks at room temperature.
Use of the kit will 1. considerably simplify resistance detection 2. ensure that chemical insecticides used to control pe.;;s and disease vectors are effective and hence save lives, cost and man: ower 3. allow constant and regular monitoring of the susceptibility of target insects and 4. assist in the design of new control agents, counter-measures and understanding of the mode of action of insecticides.
DESCRIPTION OF PREFERRED EMBODIMEI'r Eight containers were prepared containing, respectively 10 nl of potassium phosphate buffer (pH 6.8) x 2, 1 ml of acetone, 7.5 mg acetylctioline iodide, 9 ml of potassium phosphate buffer, 1.3 mg of 5,5-dithio-bis-(2-r itrobenzoic acid), 1mg propoxur as a solution of 25 pg in 10 ml 70% ethanol and 4 ml of potassium phosphate buffer.
The acetone is poured into the acetylcholine iodide and mi> d well to form a solution, which is then added to the 9 m buffer. O ne oft h 1 0 m buffers is poured into the 5,5-dithlo-bis-(2-nitrobenzoic acid) and mixed well to form a solution, 1.8 ml of the acetyicholine iodide solution is mixed v th the 4 ml buffer and 0.2 ml is then added and mixed well.
A single mosquito is homogenized in one drop of the buffer In ;an Eppendorftube using a pestle and further diluted with buffer to the 0.5 ml mar'. on the Eppendorf tube. After the homogenisate has stood for 5 minutes to al ow debris to settle one drop of the homogenisate is dropped into each well of a four well microassay plate. One drop of the undiluted acetylcholine iodid solution and one drop of the 5,5-dithio-bis-(2-nitrobenzoic acid) solution are cl-opped into two of the wells. One drop of the propoxur solution is dropped Into the other two wells.
One drop of the 5,5-dithio-bis-(2-nitrobenzoic acid) solution i; then dropped Into these two wells. The plate was incubated for 30 minutes and then the colour in the samples in the two sets of wells was assessed. If the. yellow colour in the P MPDDMA ~sptlf 11-07593% d.-5Nc.12008 00 O second pair of wells containing propoxur solution is lighter than that in the first pair of wells the mosquito is susceptible to acetylcholine esterase and therefore not resistant to organophosphate and carbamate based insecticides.
00 5 Colour can be checked by eye or by scanning using an immunoassay reader at 410 nm.
00 The reference in this specification to any prior publication (or information derived from Sit), or to any matter which is known, is not, and should not be taken as, an acknowledgement or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Claims (2)
- 6- The claims defining the present invention are as follows: 1 A method for checking for sensitivity to acetylcholine esterase using a single insect, which comprises the steps, in any order, of homogenizing a single insect in 00 00 a neutral to acid pH phosphate buffer, producing a solution of acetylcholine iodide Sin a neutral to acid pH phosphate buffered water-miscible organic solvent, O producing a solution of 5,5-dithio-bis-(2-nitrobenzoic acid) in the buffer, and N producing a solution of propoxur in the buffer, dropping the insect homogenisate into the wells of an assay plate, then dropping the acetylcholine iodide and dithio-bis-(2-nitrobenzoic acid) solutions into some of the wells and (2-nitrobenzoic acid), acetylcholine iodide and propoxur solutions into the other wells and checking by eye for a difference in the yellow coloration between the samples in the two sets of cells. 2 A method according to claim 1, which uses a test kit comprising a container containing separate containers of: I. a neutral to acid pH phosphate buffer II. acetylcholine iodide Ill. 5,5-dithio-bis-(2-nitrobenzoic acid) and IV propoxur V. a water-miscible organic solvent 3 A method according to claim 1 or claim 2, wherein the buffer is a pH 6 to 7 buffer. 4 A method according to claim 3, wherein the buffer is a pH 6.8 buffer. A method according to claim 1 or claim 2, wherein the buffer is potassium phosphate. P:\WPDOCS\AGs\lpfication\775S> doc.5/fn)1lMllX 00 -7- O O 6 A method according to any one of claims 1 to 5, wherein the propoxur is used as solution of 5 to 25 mg/1 in ethanol. 7 A method according to any one of claims 1 to 6, wherein the propoxur and 00 5 acetylcholine iodide solutions are pre-mixed. 00 c 8 A test kit when used for determining susceptibity of acetylcholine esterase in Sinsects using a single insect, including a container containing separate containers of: I. a neutral to acid pH phosphate buffer II. acetylcholine iodide III. 5,5-dithio-bis-(2-nitrobenzoic acid) and IV. propoxur V. a water-miscible organic solvent. 9 A test kit according to claim 8, wherein the buffer is a pH 6 to 7 buffer. A test kit according to claim 8 or claim 9, wherein the buffer is a pH 6.8 buffer. 11 A test kit according to claim 1, wherein the buffer is potassium phosphate. 12 A test kit according to any one of claims 8 to 11, wherein the propoxur is used as solution of 5 to 25 mg/l in ethanol. 13 A test kit according to any one of claims 8 to 12, which also contains equipment for carrying out the test. 14 A test kit according to claim 13, which contains test tubes, a pipette and a micro- assay plate. P %WPDOCSIAG ,fmirjlclin 7
- 758390.doc.5AJ2COJB 00 O 0-8- O 15 A method for checking for sensitivity to acetylcholine esterase substantially as Sherein described. 16 A test kit for determining susceptibility of acetylcholine esterase in insects 00 5 substantially as herein described. 00
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| MYPI20022441 | 2002-11-26 | ||
| MYPI200224415 | 2002-11-26 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2002318877A1 AU2002318877A1 (en) | 2004-06-10 |
| AU2002318877B2 true AU2002318877B2 (en) | 2008-11-06 |
Family
ID=34309593
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2002318877A Ceased AU2002318877B2 (en) | 2002-11-26 | 2002-12-18 | Method and test kit for the detection of insecticide resistance in insect vectors |
| AU2002318878A Ceased AU2002318878B2 (en) | 2002-11-26 | 2002-12-18 | Method and test kit for the detection of insecticide resistance in insect vectors |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2002318878A Ceased AU2002318878B2 (en) | 2002-11-26 | 2002-12-18 | Method and test kit for the detection of insecticide resistance in insect vectors |
Country Status (1)
| Country | Link |
|---|---|
| AU (2) | AU2002318877B2 (en) |
-
2002
- 2002-12-18 AU AU2002318877A patent/AU2002318877B2/en not_active Ceased
- 2002-12-18 AU AU2002318878A patent/AU2002318878B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| AU2002318877A1 (en) | 2004-06-10 |
| AU2002318878A1 (en) | 2004-06-10 |
| AU2002318878B2 (en) | 2008-08-07 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGA | Letters patent sealed or granted (standard patent) | ||
| MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |