AU2002318877B2 - Method and test kit for the detection of insecticide resistance in insect vectors - Google Patents

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.

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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)

1. 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
2. 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