Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method

Abstract

Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity. In this study, we used a conserved sequence in the 200- to 300-fold repetitive 529 bp gene of Toxoplasma gondii to design primers for LAMP test. Detection limit of T. gondii LAMP assay with the primers is 1 pg/μL of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. Furthermore, LAMP and conventional PCR methods were applied for amplification of the T. gondii DNA extracted from the lymph nodes taken from pigs which were suspected to be Toxoplasma infection. As a result, 76.9% (70/91) and 85.7% (78/91) of the samples were positive on PCR and LAMP analyzes, respectively. Therefore, the LAMP has a potential to be applied as an alternative molecular diagnostic tool for detection of T. gondii infection from veterinary samples. This is the first study, which applies the LAMP method to diagnose Toxoplasma from veterinary samples.

Introduction

Toxoplasma gondii is an obligate intracellular protozoan parasite belonging to the phylum Apicomplexa, subclass Coccidia. Infection by parasite can be acquired by eating raw meat containing tissue cysts, or food and water contaminated by oocysts (Dubey, 2004). The clinical manifestation is usually benign in immunocompetent hosts, but can be life-threatening in an immunocompromised patients (Luft and Remington, 1985).

Diagnosis of toxoplasmosis can be achieved by a number of different methods. Serological methods like indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA) are used to detect antibodies against T. gondii (Jannuzzi et al., 1981, Mondesire et al., 1981). Immunohistochemical staining and polymerase chain reaction (PCR) methods are also used for specific diagnosis of T. gondii infection (Dubey and Beattie, 1988). The modified agglutination test (MAT), immunochromatographic test (ICT) and the real-time PCR have also been developed as diagnostic methods for T. gondii infection (Huang et al., 2004, Edvinsson et al., 2006, Mainar-Jaime and Barberán, 2007). Despite these advances, diagnosis of T. gondii infection remains unsatisfactory because serological methods cannot differentiate past and present infections, whilst PCR methods are limited by need for expensive equipment.

A novel nucleic acid amplification method, loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, simplicity and rapidity under isothermal conditions has recently been developed (Notomi et al., 2000). Since the advent of the LAMP, it has been developed for detection of many viral, bacterial, protozoan and fungal diseases (Kuboki et al., 2003, Maruyama et al., 2003, Endo et al., 2004, Okafuji et al., 2005). Recently, this method of novel gene amplification has been developed and used successfully for the diagnosis of parasitic infections, such as malaria, trypanosomiasis, theileriosis and babesiosis (Poon et al., 2006, Alhassan et al., 2007a, Alhassan et al., 2007b, Iseki et al., 2007, Thekisoe et al., 2007a, Thekisoe et al., 2007b, Thekisoe et al., 2007c, Njiru et al., 2008). As a result, LAMP has been reported to be a highly sensitive and specific method for the detection of parasitic infections. Moreover, LAMP is an attractive diagnostic method in resource poor countries where facilities are minimal as a rapid test, independent of specialized heating equipments.

The 200- to 300-fold repetitive 529 bp fragment of T. gondii have been utilized for the development of a very sensitive and specific PCR for diagnostic purposes, and in a quantitative competitive-PCR for the evaluation of cyst numbers in the brains of chronically infected mice (Homan et al., 2000, Edvinsson et al., 2006). In this study, we used the conserved region in the 529 bp gene to design LAMP primers, for sensitive and specific detection of T. gondii. Furthermore, we evaluated the detection sensitivity of T. gondii LAMP in comparison with the conventional PCR on swine DNA samples.