Comparison of a Nucleic Acid Sequence-based Amplification (NASBA) and real-time reverse transcriptase PCR methods for detection of Toxoplasma gondii in rat blood samples

Introduction

Toxoplasma gondii is an obligatory intracellular organism that infects one-third of the world population and is an opportunistic pathogenic parasite, causing toxoplasmosis (Montoya and Liesenfeld, 2004). Toxoplasmosis generally occurs by ingestion of contaminated food on undercooked meat containing tissue cysts. Neonatal or acute infections in pregnant women can give rise to congenital mental retardation, blindness and hydrocephalus (Duval et al., 2004). In immuno-compromised hosts (i.e. acquired immunodeficiency syndrome(AIDS), organ-transplanted and some cancer patients), the development of fatal Toxoplasma encephalitis is believed to be due to the transition of resting bradyzoites to rapidly replicating tachyzoites (Boyer et al., 2005). A rapid, accurate and effective diagnosis is crucial and desirable to initiate adequate treatment in these two populations.

Current diagnosis of toxoplasmosis relies on serological detection of specific anti-Toxoplasma immunoglobulins. The congenital toxoplasmosis of a fetus may remain undetected since the pregnant mother might serologically be negative during the active phase of T. gondii infection. Serological testing may fail during the active phase of T. gondii infection because the antibody titers. Moreover, serological tests may fail to detect T. gondii infection in certain immunocompromised patients due to the fact that the titers of anti-Toxoplasma antibodies may fail to rise (Calderaroet al., 2006). Therefore, molecular amplification techniques can provide highly sensitive and specific methods for the detection, identification and characterization of toxoplasmosis in these patients (Bretagne et al., 1993). However, due to the robustness of DNA, the signal is not necessarily related to the detection of viable infectious parasites. Therefore, there has recently been interest on the use of RNA as the molecular target for viability assessment. Both mRNA and rRNA have a shorter half-life than DNA, and therefore, they should provide a better indication of viable organisms than DNA. However, if mRNA is used there must be certainty of existence of targeted mRNA for transcription under most assay conditions (Sheridan et al., 1998; Birch et al., 2001).

NASBA, first described by Kievits et al (1991) involves the simultaneous use of three enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT), RNase H, and T7 RNA polymerase, under isothermal conditions (41 °C). The final amplification product is a single-stranded RNA of opposite sense of the original target. More recently, real-time detection based on molecular beacon probes has been described (Leone et al., 1998). Furthermore, nucleic acid sequence based assay (NASBA), has been developed for the diagnosis of various infectious parasites, including Plasmodium (Schoone et al., 2000), Leishmania (Meide Van der et al., 2005) and Trypanosoma brucei (Mugasa et al., 2008).

In the present study, the NASBA was adapted to real-time molecular beacon technology for diagnosis of toxoplasmosis based on their B1 rRNAs. Molecular beacons are small, single-stranded nucleic acid, hairpin probes that brightly fluoresce when they are bound to their targets (Tyagi and Kramer, 1996).

Materials and Methods

The RH strain tachyzoites were obtained after peritoneal lavage of mice inoculated with the RH strain. Parasites collected from the mouse ascetic fluid were washed and resuspended in phosphate- buffered saline, and then centrifuged and washed twice, with centrifugation at 2000 g for 10 min. The concentration of tachyzoites was determined by phase-contrast microscopy using the counting chamber. The parasite pellets were used for RNA extraction.

Nucleic acids were extracted by the High Pure RNA Isolation kit (RNxplus) from whole blood according to the manufacturer's recommendations. Nucleic acid concentration was determined by spectrophotometry using the NanoDrop ND- 1000 device (NanoDrop Technologies, Delaware, USA) and the 260/280 and 260/230 ratios were calculated (Sambrook and Russell, 2001).

The oligonucleotide primers and probes used in this study are shown in Table 1. To the specific primers for toxoplasma were designed based on the conserved region of the B1 gene from GenBank.

Molecular beacon was designed for toxoplasma based on the probe sequence with additional sequences at either end to form the stem structure of the molecular beacon. The stability and predicted structure of the beacons were analyzed by using the European MFOLD server (http://bibiserv.- techfak.uni-bielefeld.de/mfold/). The 5' and 3'ends of the molecular beacons were labeled with the fluorescent dye fluorescein (FAM) and the nonfluorescent quencher, 4-(4'-dimethylaminophenylazo) - benzoic acid (DABCYL), respectively. Sequences of the primers and probe are given in Table 1.

The Real-time NASBA using was synthesized by in vitro transcription from a PCR product obtained by T.gondii genomic DNA and primers T7 ToxoF and ToxoR (Table 1). Reactions were performed with TaqMan PCR core reagents (Applied Biosystems-Roche Molecular Systems Inc, Branchburg, NJ, USA) and 50-μL final volume including 1× bufferII, 1.5 mM MgCl2, 200 μM dNTPs, 900 nM of each primer and 1 U AmpliTaq Gold DNA polymerase. Reactions were run in a Master Cycler Gradient device (Eppendorf AG, Hamburg, Germany), using the following program: 10 min at 95 °C; 40 cycles of 20 s at 95 °C, 30 s at 58 °C and 1 min at 72 °C; and 7 min at 72 °C. The DNA-Toxo-1730 product was quantified by spectrophotometry and analyzed by standard ethidium bromide-stained agarose gel electrophoresis (2% w/v).

Transcription was performed with 4μl 5X Buffer, 2μl DTT, 100mM,  20–40u Recombinant RNasin® Ribonuclease Inhibitor, 4μl rATP, rGTP and rUTP (2.5mM each) (prepared by mixing 1 volume deionized water with 1 volume of each of the 10mM rATP, rGTP and rUTP stocks supplied) 2.4μl 100μM rCTP (diluted from stock) 1μl linearized template DNA, 15–20u T7 RNA Polymerase.The reaction mixture is incubated at 37°C for 60 minutes RNA was analyzed by electrophoresis using denaturing conditions in an agarose/formaldehyde gel system.

Real-time NASBA on B1 rRNA T.gondii (GenBank accession number AF179871) was performed on a NucliSens EasyQ analyzer (bioMe´rieux) using the Nuclisens Basic kit for amplification according to the manufacturers manual at a KCl concentration of 80 mM. Reactions were carried out in a final volume of 20 μl. The reaction mixture was prepared by sequential addition of 10 μl of NASBA reagent primer mix containing 0.2 μM of each primer and 0.1 μM of the molecular probe, 5 μl of RNA template and 5 μl of enzyme-mix containing RNase H, T7 polymerase and AMV-reverse transcriptase. Before the addition of the enzyme mixture, the mixture was incubated at 65 °C for 2 min to uncoil secondary and tertiary structures, and then held at 41 °C for 2 min. The final mixture was incubated at 41 °C for 90 min to complete the amplification. As a negative control for each run, RNase-free water added to the NASBA reaction instead of the Toxoplasma RNA template controls. RNA from T. gondii strain RH was used as positive control. Fluorescence was measured every 120 s.

The results were considered positive when a typical logarithmic curve above the threshold line was obtained.

The primers and probe for Real-time RT-PCR were based on the same B1 rRNA sequence as used for the Real-time NASBA (Table 1). Briefly, 5 μl of extracted or diluted RNA was reverse-transcribed using200 units of M-MLV-RT (Invitrogen Canada Inc.), 5 μM of pd (N) 6 random hexamers (Amersham Biosciences), 0.25mM of each dNTPs (Promega Corporation), 2mM DTT (Invitrogen Canada Inc.) and incubated at 25 ◦C for 10 min, 37 ◦C for 50 min, followed by 5 min at 94 ◦C to stop the reaction. Complementary DNAs were then stored at −20 ◦C until further use in a two-step conventional RT-PCR or TaqMan real-time RT-PCR. Conventional PCR amplifications were performed using the same 116-bp B1 rRNA sequence as used for the Real-time NASBA described in Table 1, using an Eppendorf Mastercycler gradient system (Brinkmann Instruments Canada, Ltd., Mississauga, ON, Canada). The reaction mixtures contained 1× PCR buffer with 1.5mM MgCl2, 0.20mM dNTP, and 0.20 μM of each primer, 1 unit of Platinum Taq DNA polymerase (Invitrogen Canada Inc.) and 1μl of cDNA in a 20μl reaction. Amplification conditions were: initial denaturation at 94 ◦C for 2 min, followed by 35 cycles of denaturation at 94 ◦C for 30 s, primer annealing at 58 ◦C for 30 s and extension reaction at 72 ◦C for 45 s. A final extension was performed at 72 ◦C for 5 min. The PCR products were then analyzed by electrophoresis on 3% agarose gels (Invitrogen Canada Inc).

Fifteen Toxoplasma-free rats were injected intraperitoneally using a 23-gauge needle, with 0.2 ml of 1 × 10⁷/ml T. gondii tachyzoites of the RH strain, freshly collected from peritoneal lavage of mice. The optimum time to collect bloods is 48 hours after initial inoculation. RNA was isolated from conjunctive vein whole blood and was stored at -70 °C.

Table 1. Oligonucleotides used in the NASBA assays for T.gondii

Underlined: T7 promoter sequence.

Lowercase: stem sequences for molecular beacons.

Results