Validation of Loop-Mediated Isothermal Amplification method for Specific Detection of Camelpox Virus Genome

الموضوعات

طب

الملخص

Camelpox (CP) is a highly contagious viral disease affecting mostly young animalsand characterized by papular eruption on the skin and mucus membrane. Thecausative agent is an orthopox virus of the family poxviridae. The disease causeshigh mortality and morbidity in its singlehost species. Camelpox is wide spread inthe arid zone of Africa, the Middle East and central Asia.Loop-mediated isothermal amplification (LAMP) was optimized to amplify thehemagglutninin (HA) gene of camelpox virus (CMPV). LAMP is a novel methodof gene amplification that amplifies nucleic acid with high specificity, efficiency,and rapidity under isothermal conditions with a set of six specially designedprimers that recognize eight distinct sequences of the target. Primers used for geneamplification were designed from the camelpox Hemagglutinin gene. The wholeprocedure is very simple and rapid, and amplification could be obtained in lessthan 1 h by incubating the reaction mix in a single tube at 63°C and visualizing theproducts by agarose gel electrophoresis. Naked-eye visualization for amplificationof LAMP method was also possible through observation of a color changefollowing addition of 1 µl (1:1,000) of SYBR Green I dye to the tube. In positiveamplification, the original orange colour of the dye changes to green, which can beseen under natural light as well as under UV light (302 nm).The sensitivity and specificity of the LAMP were evaluated with PCR using sixextracted DNA samples of different poxvirus-species (camelpox, sheeppox,vaccinia and parapox virus). Both LAMP and PCR showed the same specificity asboth detected camelpox without false positive result with other pox virus species.The specificity of the LAMP was observed by the absence of any cross-reactionivwith other, closely related, members of the pox viruses species. When thesensitivity of the developed LAMP assay was compared to that of PCR, it wasfound that the LAMP demonstrated 16-fold higher sensitivity compared to PCRwith a detection limit of LAMP in serially diluted quantified camelpox DNA of0.075 ng/µl, compared to that of PCR (1.25 ng/µl). These results demonstrate thatthe LAMP-based assay is a useful tool for the rapid and sensitive diagnosis ofcamelpox. It is cost-effective, highly sensitive, and specific. The developed assayis expected to improve laboratory methods for diagnosis of this important diseasein the Sudan and in the other countries where camels are raised and subsequentlyassist in its control.

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التعريف والنوع

رقم الوثيقة

07a2bd76-cf23-4e25-a08f-a58e95e2ef90

رقم العقد

0

نوع الوسائط

Crawler

نوع المحتوى

الرسائل العلمية

صيغة المصدر

رسائل ماجيستير

نوع الملف

pdf text

أسماء الملفات

1638027_1.pdf

بيانات النشر

ألقاب المؤلفين

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اللغة

English

المصدر والدورية

اسم المصدر

Validation of Loop-Mediated Isothermal Amplification method for Specific Detection of Camelpox Virus Genome

المحتوى والصفحات

عدد الصفحات

0

إشراف وإعداد

الإشراف

عبدالمالك خلف الله

الإعداد

رشا عوض

الاقتباسات الببليوغرافية