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Toxoplasma gondii is a single-celled eukaryotic protozoan parasite capable of infecting all warm-blooded animals including humans, avian, livestock and aquatic mammals (Dubey et al., 2007a, Dubey et al., 2007b, Nissapatorn et al., 2004). Felids play the role of the definitive host for the parasite as only they can produce the environmentally resistant oocytes which become highly infectious when it sporulates (Herrmann et al., 2010). Infection happens when a warm-blooded organism comes into direct contact with tainted feces or tail. An infection also takes place when one unwarily ingests water or undercooked meat laced with T. gondii (Petersen, 2007). The other pathway that has been identified for transmission is through pregnant mothers passing on the infection to their fetus congenitally (Jones et al., 2003).
Although nearly one-third of the world human population is chronically infected with T. gondii, it is usually asymptomatic in healthy individuals (Quan et al., 2008). However, in an immunocompromised individual, complications due to the infection can be fatal as it can cause cervical or occipital lymphadenopathy, ocular toxoplasmosis, congenital toxoplasmosis, encephalitis and even schizophrenia (Petersen, 2007, Jones-Brando et al., 2003). It is also a proven cause abortion, stillbirth, infertility and neonatal mortality in farm animals world-wide resulting in huge loss of revenue (Rajamanickam et al., 1990).
Thus it is important to be aware of the dangers of the parasite and try to understand it further by studying it extensively. To carry out a study and detect strains of T. gondii, various methods are available such as PCR, DNA sequencing, ASO probes, DNA microarrays and hybridization. But for this study, the chosen method is conventional PCR, as it is proven to have greater sensitivity, ability to analyze multiple samples rapidly, relatively low cost and its ability to be discriminate between species and strains given that suitable primers are selected for the analysis (Rochelle et al., 1997). Therefore, for this research purpose, eight different markers that are up to the task have been identified and optimized accordingly.
Materials and Method
DNA Fragment Template
The RH strain of T. gondii which were used in this optimization was obtained as a gift from Prof. Rahmah Noordin (INFORMM, Penang). The parasite's DNA was isolated from pellets, which were initially stored in -80 °C freezer. Isolation of the DNA template from the pellets was done by using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), according to the protocol suggested by the manufacturer. The elution was done by using 100 µl of AE Buffer. The products were tested for quality by using the NanoPhotometer (Implen, Munich, Germany) as suggested in the manufacturer's protocol and also by running them on 1% agarose gel stained with ethidium bromide. The samples are rejected if RNA bands were visible in the electrophoresis results.
The chosen markers for the task are as listed in Table 1. The special case here is the marker SAG2 whereby the locus has two polymorphic sites at 3´ and 5´ ends for Type II and Type III of the parasite (Howe et al., 1997). So the amplification of this particular locus was performed separately at either end (Behzadi et al., 2003, Ferreira et al., 2008). All the other markers were chosen as they are commonly used for identifying T. gondii from various isolates (de Melo Ferreira et al., 2006, Khan et al., 2005, Su et al., 2006). The sequence of each primer was reconfirmed by referring to Toxoplasma Genome Map Database. The PCR primers were synthesized by a commercial laboratory and were of PCR grade (1st Base, Shah Alam, Malaysia).
The variables investigated for the optimization of the eight markers includes magnesium chloride (MgCl2) concentration, annealing temperature, PCR additives and sensitivity (Rochelle et al., 1997). Theoretical analysis of each forward and reverse primer was carried out using a primer analyzing and designing software (PearlPrimer, v1.1.19). With the software a theoretical annealing temperature was obtained for each primer and the test to decide on the optimal temperature was carried out within a ±5 °C range of the theoretical temperature. If the results were not satisfying than the temperature range was shifted accordingly. The initial annealing temperature range for each marker optimization is shown in Table 2. From studying previous literatures, the trend for the optimum concentration of MgCl2 used was identified as 1.5 mM. Barring that in mind, each marker was optimized for MgCl2 concentration between 1.5 mM and 2.0 mM range.
The basic content of the PCR tube was always; 2.0 µl of 10 X Taq buffer, 0.4 µl of dNTP, 0.2 µl of each forward and reverse primer as well as the Taq DNA Polymerase. Each tube also contained a constant amount of DNA template, which was set at 1 µl. The MgCl2 concentration varied between 1.20 - 1.60 µl. The PCR grade Milli-Q water's (Millipore Corp., Massachusetts, USA) volume, ranged between 14.40 - 14.80 µl per tube depending on the MgCl2 concentration. The choice of using the Milli-Q water was made after comparing results with commercially available PCR grade water. Each PCR tube used in this experiment had a final volume of 20 µl and was given a quick-spin in microcentrifuge prior to amplification.
The PCR amplification process was carried out in the 96-well MyCycler thermal cycler (Bio-Rad, California, USA). Although the annealing temperature varied for each primer, the other steps were carried out in constant conditions. The reaction mixture is denatured at 95 °C for 5 minutes, followed by 35 cycles of denaturing step for 30 seconds, annealing for 30 seconds and extension at 72 °C for 1 minute. Final extension incubation at 72 °C was carried out for 10 minutes before the tubes we incubated in the thermal cycler at 4 °C infinitely prior to collection.
The PCR products were observed with 1% agarose gels, stained with ethidium bromide. The gels were run at 100 V and 400 mA for an hour before being observed under Syngene InGenius L UV transilluminator (Synoptics, Cambridge, UK). Although the optimization steps were performed using Taq DNA polymerase, confirmation step for each optimized primer was done with Pfu DNA polymerase. So for the confirmation step both 10 X Taq buffer and Taq DNA polymerase were replaced with its Pfu counterparts accordingly.
Purity of DNA Fragments
The DNA fragments isolated from the pellets were estimated for their purity by using the NanoPhotometer. The ratio of A260/A280 is determined for pellets, which recorded a value of 1.865 and 1.814 accordingly. The gel image obtained as shown in Figure 1 had no RNA bands on them.
MgCl2 concentration and Annealing Temperature of Primers
The first primer optimized was 3' SAG2 where the primer was initially optimized between 55 - 65 °C. Unsatisfactory results obtained within the range prompt further tests at two different temperature ranges; 45 - 55 °C and 63 - 68 °C. The second temperature range is shown in Figure 2(A) and it clearly indicates that the band is only present at about 63 °C, and it was also best observed at 1.5 mM MgCl2 concentration. The next primer, 5' SAG2 had clearer and more consistent bands with 2.0 mM MgCl2 concentration and the band was clear at 59.9 °C as shown in Figure 2(B). BTUB was best visible with 1.5 mM MgCl2 concentration and 58.7 °C annealing temperature as depicted in Figure 2(C). Similarly, cB21-4, GRA1, GRA6 and SRS1 also had optimal MgCl2 concentration of 1.5 mM. Meanwhile, SAG3 was best observed with 2.0 mM MgCl2 concentration. On the other hand, the annealing temperature of cB21-4 was 53.7 °C, GRA1 was 61.1 °C, GRA6 was 57.8 °C, SAG3 was 66 °C, and finally SRS1 was 64.2 °C.
It is an established fact that for determination of nucleic acid concentration in solutions, the absorbance at wavelength 260 nm (A260) is typically used. The purity of nucleic DNA in a solution can be determined by obtaining the ratio of A260/A280, where a pure product will have a reading between 1.80 - 2.00 A (Kartha et al., 2007). The pellets used in this research were purified and had a reading within the given range, indicating the positive quality of the DNA fragments used in this research. Thus, we can be sure that they were clear of any protein contamination as well as RNA contamination as can be observed in Figure 1. It can be seen that the gel is free of RNA bands.
On the annealing temperature optimization front, 3' SAG2 marker proved to be the tougher one to optimize as it required tests on three different annealing temperature ranges (45 - 55 °C, 55 - 65 °C and 63 - 68 °C) before the best possible annealing temperature could be decided. The initial temperature range showed inconclusive results, while the lower temperature range gave a too many unspecific bands. The final temperature range which was tested showed that there would be no band when the temperature exceeds 63.3 °C. So the annealing temperature of 3' SAG2 was decided to be 63.0 °C as it best represented the band at about 222 base pairs as well.
Highly thermostable DNA polymerase is used in PCR reactions as it can withstand the repeated heating and cooling inherent in PCR and synthesize DNA at high temperatures. Besides, consideration of error rate also important in selecting the efficient DNA polymerase. The error rate of Pfu DNA polymerase in PCR is 2.6 x 10-6 nucleotides/cycle and lower than Taq DNA polymerase which is 2.0 x 10-4 nucleotides/cycle. Pfu DNA polymerase is the enzyme that catalyzes the template dependent polymerization of nucleotides into duplex DNA in the 5' to 3' direction. Pfu DNA polymerase also exhibits 3' to 5' exonuclease (proofreading) activity that enables the polymerase to correct nucleotide incorporation errors. However, Pfu DNA polymerase is more expensive compare to Taq DNA polymerase. Therefore, in this experiment we used Taq DNA polymerase for optimization and reconfirmation of results is done with Pfu DNA polymerase.