الكشف عن وجود الحمض النوووي للحلم و آفات الحشرات في عينات من الحبوب المخزنة منزليا بواسطة طرق البيولوجيا الجزيئية
Date
2013-05-25
Authors
لمياء يوسف أحمد الهلسه
lamia yousef ahmad alhalaseh
Journal Title
Journal ISSN
Volume Title
Publisher
AL-Quds University
جامعة القدس
جامعة القدس
Abstract
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.
Description
Keywords
الكيمياء الحيوية والاحياء الجزيئية , Biochemistry & Molecular Biology