**5. Molecular biology techniques for** *T. gondii* **detection**

The diagnosis of toxoplasmosis is usually based on the detection of antibodies by ELISA serology, agglutination assays or other methods such as Western blot immuno- or Sabin-Feldman [47] staining. However, there are times when serological or with any of the afore‐ mentioned detection methods are not possible; in these cases the techniques of molecular biology have been helpful in the diagnosis of *T. gondii*, especially the PCR technique, which has proved to be a valuable tool [2,48].

Currently different strategies for molecular diagnosis of *T. gondii* have been developed; this has meant that various methodologies are reported (both for the extraction of nucleic acids and for the detection of different amplification targets) and brings about a large variation between results from different laboratories [49]. So the aim of this chapter is to review the main methods of extraction, purification, detection and quantification of DNA from *T. gondii* in different species intended for human consumption.

#### **5.1. DNA extraction methods**

The extraction and purification of nucleic acids is the first stage of most molecular biological studies; extraction methods allow to obtain nucleic acids from various sources and then perform specific analysis by polymerase chain reaction (PCR) or its variants [49]. The quality and purity of the nucleic acids are two of the most important elements of such analysis since contaminants can interfere by inhibiting the amplification process in which PCR [50] rests. This makes clear that the importance of the process of sample preparation and DNA extraction methodology used, to have significant impact on the sensitivity of the test [50,51].

To extract the nucleic acids of the biological material, samples must be homogenized, causing cell lysis, proteins be removed by incubation with a protease and finally nucleic acids should be separated from other cellular components [50]. The ideal lysis procedure, which usually consists of a balancing techniques, must be strong enough to break the complex starting material (a fabric, for example), but gentle enough to preserve the nucleic acids. The lysis process is generally performed by physical or chemical processes, which break the bonds between the cells to facilitate interaction with lysis solutions that help release the genetic material [50, 51]. Among the conventional methods for extracting genetic material from T*. gondii* in tissue include the following procedures previous to DNA extraction.

#### *5.1.1. Mechanical homogenization with liquid nitrogen*

This process involves macerating the sample with liquid nitrogen using a mortar to obtain a fine powder. The nitrogen immediately can freeze the sample to prevent crystals formation, thus avoiding the breakdown of cell structures and the start of DNA degradation process by the action of DNase [51].

#### *5.1.2. Chemical homogenization*

In the chemical homogenization, the samples are maintained in solution at high temperatures in the presence of proteases, detergents and chaotropic agents to break the bonds between the cells or can even pierce the cell membrane. In fibrous tissue it is recommended that you cut into small pieces to facilitate their decomposition. Before starting this type of homogenization it is necessary to have information about the right amount of tissue; for rapid and complete homogenization, it is necessary to ensure the recovery of DNA and prevent degradation [51].

#### *5.1.3. Pepsin digestion*

Pepsin digestion is a method developed by Jacos et al. in 1960 and is modified by Dubey (1998) for retrieving from *T. gondii* from 50 g of muscle tissue (Dubey, 2010). The method is based on the digestion process by which the parasite is released from tissue cysts to invade its host. In this method, the homogenization of muscle tissue occurs by digestion with porcine pepsin solution, HCl, NaCl and distilled water at 37°C for 1 hour. Subsequently, a series of centrifu‐ gations allow the parasites to concentrate and be more likely to find their genetic material when purification is performed. At the end of centrifugation the product has to be buffered with bicarbonate to prevent degradation of the nucleic acids contained in the homogenate [22].

Pepsin requires acidic pH for activation; it breaks the bonds between tyrosine and phenylala‐ nine partially degraded proteins. Pepsin polypeptides of different sizes and some amino acids are obtained without degrading completely, so this procedure is commonly followed by the enzymatic action of another protease [52].

#### *5.1.4. Proteinase K*

Proteinase K is most often used because it is the widest-spectrum (degrades all proteins) protease and it is often used with buffers containing SDS and EDTA [50]. Proteinase K is a protease obtained from the fungus saprophyte *Tritirachium album* and is particularly suitable for digestion in a short time. Proteinase k acts on the carboxyl group of amino acids and is highly stable in a wide range of temperatures and pH values, having its greatest activity at elevated temperatures [53].

#### **5.2. DNA purification methods**

The DNA purification methods can be classified into two major branches: traditional protocols and by commercial kits [51].

#### *5.2.1. Conventional protocols*

They were developed in the 1950s; organic solvents used to separate proteins and DNA, once suspended in the aqueous phase by ethanol precipitation isolate [51]. In the case of *T. gondii*, more purification methodology used in the traditional methods is performed by phenol/ chloroform.

#### *5.2.2. Phenol/chloroform*

The phenol/chloroform purification is a method of liquid-liquid organic extraction consisting of separate mixtures of molecules, which is based on the difference in solubility of individual molecules in two different liquids [52,53]. The nucleic acid extraction with phenol/chloroform involves adding equal volumes of phenol/chloroform aqueous cell lysate or tissue homoge‐ nate, mixing the two phases and allowing to separate by centrifugation (Alejos et al., 2014). The phenol/chloroform method ensures the separation of liquids in two phases (organic and aqueous lower than), because chloroform is miscible with phenol due to its higher density (1.47 g/cm3) phenol [54,55].

Nucleic acids are soluble in the upper aqueous phase because of their negatively charged phosphate backbone, while proteins and lipids are separated in the organic phase [55]. Phenol causes precipitation of proteins and polymers (including carbohydrates) that are contained in the interface between the two phases (often as a white supernatant); in the case of lipids, these

**Figure 1.** DNA purification with phenol/chloroform [55].

are dissolved in the lower organic phase. The separation between the aqueous and organic phase by centrifugation allows isolation of the DNA in the aqueous phase (Karp, 2009; Soma, 2010). Subsequently the DNA is recovered from the aqueous phase with ethanol and is insoluble, causing centrifugation to precipitate it [51].

#### **5.3. DNA extraction using commercial kits**

From the 1990s were introduced to market commercial purification kits; these kits commonly used membranes or inorganic matrices to which the DNA will bind to specific conditions (Karp, 2009). Often these arrays are stacked into small columns in centrifuge tubes so that the binding steps, washing and elution can be performed efficiently by applying a centrifugal force. Some of the advantages of using commercial kits are to increase efficiency of DNA recovery, to obtain inhibitor-free extract and to decrease the time spent for purification {51,54]

The purification procedure with commercial kits can be summarized in three steps: 1) homogenization of tissues to facilitate the selective attachment of DNA to the matrix; 2) washing to remove DNA contaminants and 3) recovering the DNA from the matrix using an eluting buffer [51].

#### *5.3.1. Silica matrix*

This method is based in selectively adsorption/desorption of nucleic acids and it has proven more efficient for the recovery of pure DNA beside biological samples (i.e., blood, tissue). Silica extraction methods produce increased DNA yield while efficiently removing PCR inhibitors; those protocols usually include a small-scale silica-based spin column. The selective adsorp‐ tion/desorption occurs when, by the action of ethanol, the DNA loses its humectant layer, exposing its phosphate groups and thereby facilitating the adsorption of the molecule to the positively charged membrane. Lipids and proteins are not related to the membrane and are removed using the wash solution and a cycle of centrifugation, while the genetic material remained is bound to the matrix [51].

#### *5.3.2. Magnetic beads*

Extraction methodology using magnetic beads or a magnet that attracts magnet to separate the beads from solutions in which they are suspended is applicable. In this case, the lysis buffer solution at acidic pH, which allows to positively charge the beads, favoring DNA binding [51,53], is added.

The methodology for the extraction and purification of DNA from *T. gondii* in tissue with magnetic beads was described by Opsteegh et al. [56]. Tissue homogenization is carried out in a bag filter Stomacher400 containing a lysis buffer (100 mM Tris HCl pH 8.0, 5 mM EDTA pH 8.0, 0.2% SDS, 200mMNaCl and 40mg/L Proteinase k), by reaction using 2.5 mL of lysis buffer per gram of sample for 2 minutes at high speed. The homogenate is placed in incubation at 55°C overnight and, after the incubation, the extract obtained is homogenized again for 1 minute; 50 mL of the homogenate is transferred to a 50-mL tube and is centrifuged at 3500 g × 45 min; 12 mL of the supernatant (crude extract) is collected for use in the purification of the genetic material of *T. gondii*.

The first step in the purification process based on magnetic beads is the removal of free biotin in the sample because this method is based on the target binding sequence labeled with biotin to the magnetic beads that are coated with streptavidin (protein high affinity for biotin). To remove free biotin from the sample streptavidin sepharose should be added to allow the biotin precipitation and to form a pellet by centrifuging and 10 mL of the supernatant are finally used for purification process [56,57].

To mark the sequences of interest with biotin is needed the addition of specific primers that are marked with this molecule [57]. The bonding occurs during hybridization, so it is first necessary to denature the sample nucleic acids by heating at 95°C for 15 min and then lower the temperature to allow hybridization; if the proposed protocol is by Opsteegh et al. [56]. the hybridization temperature is 55°C for 45 min. Once labeled primers are hybridized to the target sequence, we proceed to introduce the magnetic beads in the sample and proceed to incubate at room temperature for 60 minutes to allow binding sequences labeled with magnetic beads. They are separated from the beads using a magnet and then the DNA of the beads was recovered by washing with buffer B & W (Binding & Washing) provided in the kit extraction and finally the DNA recovered is resuspended in distilled water. Magnetic capture process is presented in Figure 2.

**Figure 2.** DNA purification from tissue employing magnetic beads [56].

Today there are also cases that implement business purification methods with organic solvents. Usually these methods are based on the phenol/chloroform by adding guanidine or any other part that improves the extraction and purification of nucleic acids. Examples of these tradi‐ tional methods are converted to commercial kits extraction and extraction with TRIzol® DNAzol® [58,59]. These commercial kits based home methods are usually cheaper than commercial kits based on inorganic matrices, although purification performance is very similar in some cases, there is a risk of contamination of samples with phenol and inhibition of amplification in the PCR process [60]. Another important factor to consider is security, since this type of extraction involves the use of corrosive and irritant substances and therefore requires some experience to handle; accidental contact with the reagents that can burn skin and vapors inhalation could cause damage to the respiratory system, in both cases, medical assistance is required. Table 1 presents the main commercial kits used for the extraction and purification of DNA from *T. gondii*.


**Table 1.** Leading commercial kits used for DNA extraction and purification from different tissues for *T. gondii* detection
