Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi

Abstract

A loop-mediated isothermal amplification (LAMP) assay was developed for the diagnosis of Theileria lestoquardi infection. The primers were designed based on the clone-5 sequence of T. lestoquardi. The specificity and sensitivity of the assay were established. Analysis of the specificity showed that the selected LAMP primers amplified the target sequence from T. lestoquardi DNA successfully, while no amplification was seen with DNA from Theileria annulata, Theileria ovis, Babesia ovis, Anaplasma ovis, or ovine genomic DNA. The specificity of the LAMP product was further confirmed by restriction digestion and sequencing. The sensitivity of the LAMP assay was analyzed in comparison to PCR resulting in a detection limit of 10 fg/μl of plasmid DNA containing the clone-5 sequence. The suitability for utilizing the LAMP assay in the field for the diagnosis of T. lestoquardi infection was tested on 100 field samples collected in Sudan and compared with results obtained by PCR. The relative specificity and sensitivity of the established LAMP assay was determined to be 92.1% and 87.5%, respectively, indicating that it may be regarded as an alternative molecular diagnostic tool to PCR which could be used for epidemiological surveys on T. lestoquardi infection.

Variation in susceptibility of three Sudanese sheep ecotypes to natural infection with Theileria lestoquardi

“…The LAMP primers and the procedure followed in reaction condition and the detection of the reaction products were essentially as described by Salih et al (2012).…”

Calpains: Potential Targets for Alternative Chemotherapeutic Intervention Against Human Pathogenic Trypanosomatids

“…In this sense, when the effects of pepstatin A, a powerful aspartic peptidase inhibitor, on T. cruzi were explored, the proliferation of epimastigote forms was arrested and significant morphological alterations were found. In addition, pepstatin A induced an increase of 54% and 98%, respectively, in the surface expression of gp63- and calpain-related molecules in epimastigotes, but not in the cruzipain level [ 68 ].…”

Theileria annulata DNA’sının loop aracılı izotermal yöntemle (LAMP) çoğaltılması

“…Ancak, bu yolla belirlenen duyarlılıklar kullanılan hedef DNA bölgesi, primer çiftleri, reaksiyon koşul-ları ve ortamda bulunan hedef DNA miktarına bağlı olarak farklılıklar gösterebilmektedir. LAMP'ın, Trypanosoma brucei gambiense'nin mikroskobik ve PZR metotlarıyla teşhisine (13), T.equi (32) ve sığır babesiosis (10) ile koyunlarda hastalık oluşturan Theileria ve Babesia soyundaki kan parazitlerinin (8,27) belirlenmesinde PZR'ye göre, B. gibsoni'nin tespitinde (17) ise nested PZR'ye göre daha duyarlı olduğu bildirilmiştir. Bunun yanında, köpeklerde görülen B. gibsoni'nin (9) ve equine piroplasmosis etkenlerinin tespitinde kullanılan LAMP yöntemi (3) ile PZR eşit duyarlılıkta hastalık etkenlerini tespit edebilmiştir.…”

“…LAMP yöntemi son yıllarda, tek nükleotid polimorfizmlerinin belirlenmesine dayalı genetik çalışmalar ile viral, bakteriyel ve paraziter etkenlerin tanısal amaçlı belirlenmesinde giderek artan oranda kullanılmaya başlan-mıştır. Paraziter kökenli hastalıklardan equine piroplasmosis (2,3,32), tropikal theileriosis (15,28), East Cost Fever (ECF) (29) ile sığır babesiosis (10,14) etkenlerinin ve koyunlarda hastalık oluşturan Theileria ve Babesia soyundaki kan parazitlerinin (8,16,27), Giardia duodenalis (26) ve Cryptosporidium türlerinin (5,12), Trypanosoma brucei gambiense'nin (13), köpeklerde B.gibsoni (17,9) ve B.canis (1,19) ile ara konak sivrisineklerdeki Plasmodium spp. 'lerin (4) teşhisi amacıyla LAMP yön-temi kullanılarak birçok çalışma yapılmıştır.…”