**6. Methods for molecular detection of** *T. gondii* **in meat for consumption**

The presence of *T. gondii* in biological samples can be diagnosed by molecular techniques aimed at detecting its genetic material [74,85] (Switaj et al., 2005; Garcia et al., 2006). A specific fragment of the parasite genome can be amplified by PCR to visualize on a polyacrylamide or agarose gel, to stain, to sequence directly in real time. The sensitivity and specificity of PCRbased techniques rely on an appropriate method of purification of genetic material from the samples, the characteristics of the DNA sequences chosen for amplification and the parameters of the amplification reaction [50,85]. Various amplification methods for *T. gondii* will be explained below.

#### **6.1. Conventional PCR (endpoint)**

The PCR is a molecular biology technique developed by Dr. Kary B. Mullis in 1985 [54,86]. The impact of his discovery was such that Dr. Mullis received the Nobel Prize for Chemistry in 1993 (Welch, 2012). PCR is a technique "*in vitro*" used to amplify enzymatically a specific region of DNA located between two regions of DNA whose sequence is known [86]. PCR simulates what happens during cell replication taking advantage of features of the chemical structure and semiconservative DNA replication [50].

DNA is a polymer formed from two complementary strands anti-parallel, each chain consists of nucleotide units which in turn are comprised of a nitrogenous base (adenine = A, guanine = G, cytokine = C and thymine = T) attached to sugar deoxyribose and a triphosphate group [50,87]. To replicate the DNA separates its two complementary strands, serving each mold or template for the "de novo synthesis" of its complementary strand, the specificity of pairing of the nitrogenated bases (TA, CG) to obtain two identical DNA molecules, each consisting of an original and a new chain. The enzyme that performs this process is called DNA polymerase [54,87]. PCR synthesis of new DNA strands is performed by mixing: containing DNA or fragments to be amplified; polymerase; primers (DNA fragment of 15 to 30 nucleotides flanking the region to be amplified and to provide the free 3 'end to initiate transcription); deoxynucleotides (dNTPs); magnesium chloride (MgCl2) or other cofactor necessary to work polymerase [86]. Generally, the PCR begins with denaturation or separation of the double helix of DNA by heating the sample at a temperature between 94 and 96°C to break the hydrogen bonds that bind them, so as each string is a template for synthesis A new complementary strand of DNA [88].

Once separated the chains of DNA primers (initiators or primers) are aligned in complemen‐ tary-specific sites of the single strands in the region to be amplified; for this to happen it is necessary to lower the temperature between 40 and 60°C, allowing binding (hybridization or alignment) of the primers. Finally, a new strand is synthesized in the 5′ to 3′ for which the temperature is increased, generally at 72°C, which is the optimum temperature to work Taq polymerase [89]. These three stages — 1) denaturation, 2) hybridization and 3) elongation of DNA — are repeated successively in each new cycle and amplified the region of interest of the two complementary strands [86] simultaneously. The essential equipment for the process to take place is the thermal cycler, which has a heating pad where each reaction is placed and where temperature changes are accurate and can be pre-programmed in three stages by several cycles [50, 86].

Detecting the PCR product is usually accomplished by electrophoresis. Separation matrices (agarose, polyacrylamide) at various concentrations are used depending on the size of the amplification product and the resolution desired. The posterior viewing can be done with ethidium bromide with a UV lamp, silver staining, fluorescence or radioactivity light. The sizes of the PCR products are determined by comparing them with markers containing DNA fragments of known size, which are run in a gel with PCR products.

#### **6.2. Nested PCR**

Nested PCR (nested) consists of two successive processes of amplification, using the product of the first amplification as template for the second [2]. In the second amplification primers used should be different from the first amplification and are targeted to amplify a smaller fraction contained within the product of the first PCR [90]. This methodology increases both the sensitivity and specificity of the test. Furthermore, the risk of contamination increases significantly due to the increased amount of amplification products and work steps involved [91]. In this type of test validation it is always recommended by both negative and positive controls, ensuring that the positive controls are highly diluted to avoid contamination of the samples [90].

#### **6.3. PCR-LAMP**

This is a variant of PCR developed for parasites of the phylum Apicomplexa, among which is *T. gondii*. The amplification method LAMP (Loop-mediated isothermal amplification) is based on a displacement auto cyclic reaction chain using a set of four oligonucleotides (primers) which recognize six sequences within the genomic DNA target region and form a loop structured amplicon, the polymerase that performs this function is Bst polymerase having activity displacement [92,93].

#### **6.4. Real-time PCR (qPCR)**

Real-time PCR is a technique used to quantify specific nucleic acid sequences in a sample of interest. The assay is based on generating a fluorescent signal that is directly proportional to the amount of target DNA. Real-time PCR is able to monitor the fluorescence emission that occurs during the reaction progress, so is said to be in real time [94]. Among the major fluorophores used for determination of *T. gondii* load are as follows:

#### *6.4.1. SYBR Green™*

It is an intercalator that binds to dsDNA resulting fluorescence increased with increasing the amount of PCR product. An important aspect to consider is that the SYBR Green™ can also join primer dimers and nonspecific amplification products, resulting in an overestimation of the concentration of target DNA. The detection of DNA of *T. gondii* using SYBR Green it could be useful mainly during experimental infections. It has been used for monitoring parasitemia loads in animal models, however for clinical diagnosis or monitoring any other natural infection (i.e., in animals for consumption) the performance it is not enough sensitive to detect the presence of low amount of the target in the sample.

#### *6.4.2. TaqMan™*

TaqMan probes are hydrolysis probes which allow increase the specificity of quantitative PCR. They have attached a reporter (a fluorophore) and a quencher. When both (fluorophore and the quencher) are in proximity, the reporter emits no signal, however, when the probe hybridizes to the sequence of interest during PCR, the endonuclease activity of Taq polymerase to short photochromic other reporter probe, allowing emission of a fluorescent signal. The reporter fluorescent signal is cumulative in each of the subsequent cycles [79].
