الكشف عن وجود الحمض النوووي للحلم و آفات الحشرات في عينات من الحبوب المخزنة منزليا بواسطة طرق البيولوجيا الجزيئية
detection of the presence of insect pests and mite DNA in samples of home stored grains by molecular techniques
لمياء يوسف أحمد الهلسه
lamia yousef ahmad alhalaseh
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Home stored grains such as wheat, rice, lentils, corn...etc, are usually exposed to contamination with insect pests. Over 60 species of insects infest stored grains where Indian meal moth, flour beetles, saw-toothed grain beetles and granary weevil are the most common. These pests are economically important and are responsible of millions of dollars loss because contamination by these pests reduces grains quality and therefore discarding them. They may also cause several health problems including allergies and gastrointestinal disorders. Insect pests are classified as primary and secondary pests. The primary pests present a bigger problem than secondary pests because they infest grain kernel; feed upon them and reproduce on it leading to major damage of the whole sound grain, while secondary pests feed on grains damaged by primary pests because they are not capable to penetrate grain kernel. The global spreading of these insects occurs as a result of word wide cereal distribution. Infestation might occur during storage, shipping and transportation. Control managements of these insect infestations can be achieved either by chemical (fumigation), physical (thermal control, inert dust, ionizing radiation, light and sound control) or biological treatments (pheromones, growth regulators, microbial control and plant extracts). There are several methods for detection of insect pests in grains. Traditional detection techniques include staining kernels, density separation, uric acid analysis, acoustical sensors, x-ray, near infrared spectroscopy (NIR) and enzyme linked immune-sorbent assay (ELISA). Problems encountered with these methods are that they are laborious, expensive and not sensitive to detect insect contamination at the egg and larvae stages. Therefore, newer methods are needed forrapid and sensitive detection. One obvious approach is to develop a molecular biology technique that utilizing genetic information of the different insects for amplification of specific target gene sequences by polymerase chain reaction [PCR] and real time PCR for that purpose. In this study, used a number of infested grain samples were used to isolate larvae and adult insects from them which serve as positive controls in our work. The isolated insects were subjected to DNA extraction, PCR amplification of defined regions in the cytochrome oxidase gene followed by sequencing to identify each pest species. The sequences were identified according using BLAST generated comparison to the original gene sequence obtained from GeneBank. The sequences of the gene from the differentinsects were aligned to design three sets of primers specific for insect mitochondrial cytochrome oxidase subunit I gene. Two primer sets, COI-long1 and COI-long2 for general pest species and COI-mite for the detection of mites. The designed primers were tested for their specificity and sensitivity. A problem was encountered with grain swelling after they were mixed with the aqueous solutions to collect the contaminating insects. This problem was solved by developing and adapting two different methods for grain treatment before DNA extraction using a centrifugation washing method or filtration washing method with the different sample size including either 10 or 50 grams respectively. For PCR optimization, the original DNA sample, 1:10 and 1:100 sample dilution were tested which indicated the best and sample dilution to use was 1:10.. The suitability of PCR primers and DNA extraction methods was evaluated on eleven samples of commercial grains in six separate PCR reactions utilizing each primer set with the two extraction methods. The detection sensitivity varied between the different primers used and extraction method where superiority with COI-long1 primer compared to the COIlong2 primer and the filtration washing method was more efficient over centrifugation washing method giving the pest combination is COI-long1 with filtration washing method.