Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B

https://doi.org/10.1016/j.exppara.2010.01.017Get rights and content

Abstract

Camel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (T. evansi type B) has been identified. The prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20–25 min at 63 °C using a real time PCR machine. Restriction enzyme AluI digestion of the amplicon gave the predicted 83 bp and 89 bp sized bands and the LAMP product melt curves showed consistent melting temperature (Tm) of ∼89 °C. The assay analytical sensitivity is ∼0.1 tryps/ml while that of classical PCR test targeting the same gene is ∼10 tryps/ml. There was a 100% agreement in detection of the LAMP amplification product in real time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas.

Introduction

Trypanosoma evansi is undoubtedly one of the most widespread pathogenic trypanosomes in the world, naturally infecting a variety of both wild and domestic animals. This success is attributed to its transmissibility by biting flies. Recently, a case of T. evansi infection in a human was confirmed in India, raising concerns of the emergence of human infective strains in Asia (WHO, 2005, Joshi et al., 2005, Powar et al., 2006). Unlike most pathogenic trypanosomes that have consistently maintained their endemic foci and hosts, T. evansi has been emerging in non-endemic areas and infecting new hosts. Outbreaks have been recorded for the first time in the endangered Himalayan bear (Muhammad et al., 2007), a farm in metropolitan France (Desquesnes et al., 2008) and in mainland Spain (Tamarit et al., 2009). This phenomenon can be expected to increase with the prevailing global climatic changes that may gradually lead to increases in arthropod vectors and vector borne diseases. Therefore development of new and improved diagnostic assays for T. evansi is a priority.

The majority of the T. evansi stocks isolated in endemic regions possess the RoTat 1.2 VSG gene. Whilst no detailed studies have been done to correlate the presence of the RoTat 1.2 VSG gene and minicircle types in T. evansi, it may be assumed that most RoTat 1.2 positive isolates possess the most common T. evansi type A minicircles within their kinetoplast DNA (Songa et al., 1990, Ou et al., 1991, Lun et al., 1992) or are natural dyskinetoplastic (altered or lack kDNA) (Schnaufer et al., 2002). A second group of T. evansi stocks isolated in Kenya are devoid of VSG RoTat 1.2 but possess a VSG named JN 2118Hu (Ngaira et al., 2005). These isolates have been shown to possess a rare minicircle type B (Borst et al., 1987, Njiru et al., 2006) and are referred to as non-RoTat 1.2 (Ngaira et al., 2005). The use of term non-RoTat 1.2 in reference to VSG JN 2118Hu isolates needs to be applied with caution since other T. evansi that are devoid of both VSG RoTat 1.2 and VSG JN 2118Hu are not necessarily absent. The T. evansi type B isolates reveal different isoenzyme patterns (Gibson et al., 1983) and minisatellite and microsatellite markers (Njiru et al., 2007a: Njiru et al., 2007b) as compared to T. evansi type A isolates. Field studies carried out in Kenya suggest that T. evansi type B is not only pathogenic to camels but may also be widespread in camel keeping regions of Kenya (Ngaira et al., 2004, Njiru et al., 2006), and its detection is likely hindered by the lack of a specific and sensitive diagnostic test.

Definitive diagnosis of surra can be achieved through demonstration of parasites by microscopy. However, this method is inadequate due to the characteristic low parasitaemia of T. evansi infections. Molecular tests are supposedly more sensitive but their laboratory requirements limit application in the endemic regions. Therefore field diagnosis relies on the established antibody detection test based on VSG RoTat 1.2, a card agglutination test for trypanosomiasis (CATT/T. evansi) (Songa and Hamers, 1988). Unfortunately, CATT/T. evansi cannot detect isolates devoid of RoTat 1.2 VSG such as T. evansi type B (Ngaira et al., 2004). Moreover, the presence of antibodies in the serum does not necessarily reflect an existing infection, as antibodies may persist for several months following recovery. Recently, a rapid and sensitive amplification platform for DNA called Loop-mediated Isothermal Amplification (LAMP) has been developed (Notomi et al., 2000). The strategy is robust, specific and shows tolerance to several biological products that inhibit conventional PCR (Yamada et al., 2006, Kaneko et al., 2007), meaning that template DNA extraction may not be necessary. Besides, LAMP results can be visually inspected through colour change (Poon et al., 2006, Njiru et al., 2008b) or coupling with chromatographic LFD (Nimitphak et al., 2008) which significantly reduces the assay time.

The LAMP technique has been successfully used to develop assays for both human (Njiru et al., 2008a, Njiru et al., 2008b) and animal (Thekisoe et al., 2005, Thekisoe et al., 2007) trypanosomiasis. In this study we report the development and validation of a T. evansi type B LAMP test and the comparison of chromatographic LFD format with SYBR Green I, gel electrophoresis and real time monitoring methods in detection of T. evansi LAMP product.

Section snippets

Preparation of template

Well characterised trypanosome DNA samples were used in this study as shown in Table 1. The DNA was prepared using the Qiagen DNA extraction kit (Qiagen, Victoria, Australia) or through the method of Sambrook and Russel, 2001.

Polymerase chain reaction

The PCR test reactions for RoTat 1.2 (Claes et al., 2004) and for VSG gene JN 2118Hu “T. evansi type B” (Ngaira et al., 2005) were performed according to the published conditions. After amplification, the PCR products were analysed through electrophoresis in 1.2% agarose

Results

The results for the T. evansi LAMP test are shown in Fig. 1, Fig. 2, Fig. 3 and Table 1. The best reaction results were obtained when the temperature was maintained at 63 °C. Inclusion of loop primers reduced the amplification time to 15–20 min (Fig. 1A) from 30 min and showed a 100-fold increase in analytical sensitivity (Table 3). Post-amplification analysis showed reproducible and consistent melt curves with a Tm of ∼89.0 °C (Fig. 1B) while Alu1 restriction enzyme digestion gave the predicted

Discussion

In this study we report the development of a rapid and sensitive LAMP test for T. evansi type B based on the amplification of VSG JN 2118Hu sequence (Ngaira et al., 2005). The assay is specific and shows analytical sensitivity of ∼0.1 tryps/ml in the laboratory which is equivalent to parasitaemia expected in the field camels. The assay is rapid, results being obtained within 20–25 min using a real time PCR machine (Fig. 1A), and after approximately 35 min in a normal water bath that maintains

Acknowledgments

The DNA samples used for specificity studies were initially provided by Prof. Wendy Gibson, University of Bristol, UK for RIME LAMP studies (Njiru et al., 2008a). The Asian T. evansi DNA were part of genetic studies (Njiru et al., 2007b) and provided by Dr. Simon Reid, Murdoch University while the KETRI DNA (currently, Kenya Agricultural Research Institute Trypanosomiasis Research Centre (KARI-TRC)) were part of T. evansi epidemiological study (Njiru et al., 2004). This work was funded by the

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