New methods for rapidly detecting Insect Problems

by Teresa Acklin
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   By Jim Bair and Dr. G. Barrie Kitto. Mr. Bair is director of government relations for the Millers' National Federation in the U.S. Dr. Kitto is professor of chemistry and biochemistry at the University of Texas at Austin, where he also serves as director of the Center for Biotechnology. This article was first delivered as a paper before the 1992 International Technical Conference & Exposition of the Grain Elevator and Processing Society.

   The detection of insect contamination of grain and grain products has been, and remains, a serious problem. The need for accurate, repeatable and rapid tests becomes more acute as the use of chemical pesticides is curtailed, the cost of fumigation continues to increase and government regulations become more stringent.

   The tests presently available for detecting the presence of insects or insect materials are by no means satisfactory. Many of them rely heavily on visual inspection and are, therefore, subject to considerable variation from inspector to inspector. For example, the insect-damaged kernel test (I.D.K.) provides only a very indirect measure of the presence of insects. This assay visually assesses the damage to grain kernels but can provide no direct information as to whether an insect is actually present at the time of examination or has simply been there at some time in the past. Moreover, the U.S. federal limit for this test (32 I.D.K./per 100 g) is far above that which is acceptable to grain millers. The correlation between the I.D.K. values on whole grain with the amount of insect contamination seen in milled grain products is poor.

   The simple visual examination of grain samples for the presence of live insects is likewise fraught with difficulties. First, the limit is numerically low (two live insects/1,000 g constitutes the defect action level), which raises the question of adequate sampling. Furthermore, the presence of live insects in a sample is not a reliable indicator of the amount of insect infestation within grain. Adult live insects are readily cleaned from grain samples, but it is the internal infestation that can cause major difficulties further down the transportation and storage pathway.

   In a few locations, x-ray analysis is available. While this technique offers good quantification of internal infestation, the cost of the equipment, licensing requirements and the need for skilled interpreters of the x-ray pictures has hindered widespread adoption of this approach. Similar liabilities surround most of the other insect contamination assay procedures.

   At the flour level, essentially the only quality control measure in widespread use for insect matter is the insect fragment count. This procedure has been the long-standing accepted standard, but suffers from a number of disadvantages. It is time-consuming, expensive and requires highly trained technicians. Even more important is the variability of the assay and its lack of precision as a technique to gauge the mass of insect material present in a sample. Part of these problems stems from the very nature of the test, which measures just the number of insect fragments present in a sample. A large fragment carries an equal weight in the results as does a very small fragment.

   Clearly, the insect fragment count is very dependent on the amount and type of processing that the sample has undergone. The insect fragment count is also highly dependent on whether the contamination resulted from live or dead insects. Dead insects result in many more fragments than do live ones. For example, a dead adult insect can result in 50 times as many fragments as a live larva.

   Another major problem in the field of insect detection is that there is no single technique available that can be applied to both whole grain and milled grain products. Nor is there even an assay procedure available for whole grain that provides an accurate and reliable correlation with the amount of insect contamination that is likely to turn up in the finished product.

   It was to overcome these problems that we have engaged in the development of rapid and sensitive biochemical assays for insect detection, supported by the U.S. Department of Agriculture (U.S.D.A.) and the Millers' National Federation.

   A very sensitive immunological assay has been devised for an insect muscle protein called myosin, which is present in all insects. In simple terms, the test involves the addition to an extract of a grain or flour sample of reagents that bind to the insect material and, through a series of reactions, result in a color being developed in proportion to the amount of insect contamination present. Using this type of approach, we have developed two distinct types of insect contamination assay, based on similar principles. One of these is a highly quantitative assay, designed to be used in small laboratories. The second is a much more rapid qualitative assay for use at grain receiving or loading sites. Both assays are of the type called ELISA, or enzyme-linked immunosorbent assay.

   An overview of the laboratory-style ELISA procedure is shown in Figure 1, on page 13.

   Briefly, a grain sample is mixed with an extracting fluid and blended in a typical household blender. A sample of the liquid extract is then applied to a multi-well plastic test plate. A sequence of reagents is then added to the well, resulting in color formation proportional to the amount of insect material present in the original sample. The color of the test is then read in an ELISA reader. The data either can be printed out directly and/or stored in a computer for record keeping.

   The assay is of high sensitivity, being able to readily detect as little as one insect per 50 g of grain sample, and provides an excellent linear response to the degree of insect infestation, as shown in Figure 2.

   The immunoassay has been tested with a wide variety of grain insect pests, as shown in Figure 3, and provides a positive linear response in each case.

   Because of the numerous problems with the variability of other types of insect assays, we have taken great pains to evaluate the performance of the immunoassay procedures. The ELISA assay has exceptionally good replicability. For example, 24 replicate tests of a single sample with an average absorption (color) value of 1.04 had a standard deviation of only 0.03.

   We have also been concerned about the appropriate initial size of the grain sample to be taken for analysis. Our studies have established that an effective measure of insect contamination is obtained when a 200-g grain sample is ground in a blender, and then 50 g of that sample are subsequently blended with the liquid extraction medium to provide a test sample for the ELISA assay.

   In addition to extensive laboratory testing, the reproducibility of the ELISA assay has also been evaluated using field samples with varying degrees of insect contamination. One such series of tests is illustrated in Figure 4. These samples were assayed either twice or three times over a one-week period and show little variance in assay results.

   The insect ELISA procedure can readily measure insect contamination in milled grain products, as well as in whole grain. Thus, this methodology provides, for the first time, an ability to predict, from an analysis of the whole grain, what is likely to turn up in the flour. As measured by the ELISA assay, the total insect material present in the milled grain products very closely matches that found in the grain (Figure 5).

   In other words, one could account for all of the material that was in the grain in terms of the separate milled products. A very similar distribution of insect material into flour, shorts and bran fractions was obtained regardless of the overall level of total insect contamination: 64% was found in the flour fraction, 6.5% in the shorts and 30% in the bran. This is close to the weight distribution in the sample, but not exactly so. The relative weight percentages are flour, 72%; shorts, 3%; and bran, 25%.

   Because of the ability to be able to predict the quality of the milled product from grain analysis, this allows the insect immunoassay to be used as a tool for blending wheats, prior to milling. An example of how well this can be accomplished is shown in Figure 6, on page 15, where varying proportions of relatively clean and dirty wheat (spiked with insects) were blended and subsequently tested by the ELISA procedure.

   The majority of our experimentation with the immunoassays has been carried out with a single type of wheat (hard red winter), but through the courtesy of the U.S.D.A.'s Federal Grain Inspection Service (F.G.I.S.) Technical Center at Kansas City, we have been able to test a wide spectrum of different varieties of wheat and have obtained similar responses for all the types tested.

   We have also examined the applicability of the ELISA assay to a variety of other types of grain, including barley, oats, several types of rice, rye, maize, soybeans and sorghum. Although these have not been tested as extensively as wheat, they appear to offer no major problems for testing by the immunoassay procedures.

   The materials for carrying out the laboratory-style ELISA test procedure have been put together in a kit form, and each of the components has been extensively tested for its ability to survive shipping and storage. The present configuration of the assay procedure allows for a shelf life of at least three months. The test kits have been undergoing an exhaustive evaluation at a number of the major grain companies around the United States.

   The multi-well ELISA procedure has been designed for testing multiple samples at one time with the facilities of a modest laboratory. It takes approximately five minutes to prepare an extract from each sample, and 20 assays can conveniently be carried out at one time. The ELISA procedure itself takes approximately two hours for a set of 20 samples.

   We also have been engaged in the development of species-specific insect assays. For the control of insect infestation in grain, it would often be highly desirable to know not just the total degree of insect contamination, but also the relative amounts of different infesting species. With adequate training, it is not particularly difficult to distinguish the adult forms of many of the major grain pests. However, the question of species identification becomes a great deal more difficult if the infestation consists of hidden insects, as is the case of typical grain in storage.

   Identification of the infesting species requires not just careful dissection of the infested grains, but many of the larval stages, which lack the simple visual cues that aid species identification with adult insects. Species identification would provide a rationale for more appropriate selection and timing of insecticide and fumigant treatment.

   Moreover, the availability of insect-specific assays would greatly facilitate the utilization of beneficial insects for pest control. Working with our colleagues at the U.S.D.A. laboratories at Madison, Wisconsin, under the direction of Dr. Wendell Burkholder, we have developed several species-specific assays for both grain pests and for beneficial insects. An example of the use of such species-specific assays is shown in Figure 7, on page 16. In this case, the myosin assay was used to evaluate the total degree of infestation, while the species-specific assay allowed the determination of what proportion of this total was due to granary weevils.

   We recognize that there are many instances where it would be desirable to have an alternate form of the immunological test for a much more rapid but more qualitative assessment of insect contamination. Such an assay could be used at locations such as grain elevators, where it could be used to rapidly inspect incoming truckloads or railroad car lots of grain, and at shipping locations, where rapid checks could be made on grain during the loading or unloading process.

   We have investigated several potential modes for carrying out such rapid assay procedures, including an examination of flow-through devices, dipsticks and membrane assays. The most promising of the techniques that we have been assessing is illustrated in Figure 8, on page 16.

   In this case, the antibody used for the binding of insect material in a sample is coated onto a small plastic bead that is held inside a plastic pipet tip. The sample to be tested is sucked into the pipet tip allowing any insect material to couple to the bead. The amount of insect material present is then determined by subsequently drawing reagents into the pipet tip, whereupon color develops in proportion to the amount of insect matter present. The amount of color generated can be assessed either using a simple colorimeter or, more qualitatively, by comparison with a set of printed color samples.

   This type of approach is well-suited to the analysis of a small number of test samples, or even a single sample at one time. It has the potential to allow assay results to be obtained within a period of 15 minutes or less. This rapid assay provides the same degree of sensitivity as the traditional ELISA procedure.

   In summary, the insect ELISA procedures allow for accurate measurement of insect contamination in a broad variety of whole grain and milled grain products. The tests have excellent sensitivity and high reliability. They can correlate incoming grain infestation to milled product contamination and are rapid and of relatively low cost. Only a very short training period is required. Collaborative multi-laboratory trials of these procedures are currently under way with the American Association of Official Analytical Chemists and with the F.G.I.S., with the anticipation that such assays will become recognized standards in the near future.