**7. Determination of parasite load in meat by qPCR**

#### **7.1. Absolute quantification**

Absolute quantitation is a quantization strategy based on comparison of the test samples against a standard curve created from a template of known concentration (Sivaganesan et al., 2010). This template of known concentration is used to make serial dilutions and generate a curve from the CT values (threshold cycle) obtained for each concentration. The curve can interpolate directly the CT values of the test samples and get your concentration by the equation *<sup>Q</sup>* =10( *CT* <sup>−</sup> *<sup>b</sup> <sup>m</sup>* ) where: Q = amount shown, CT = amplification cycle, b = insertion axis "and" m = gradient of the line. In traditional protocols creating absolute quantification standard curve in each qPCR experiment conducted it suggested, however, can also be employed master calibration curve generated from multiple experiments qPCR [95]. The absolute quantification method is conceptually easy, however, requires a reliable source as a template of known concentration.

#### **7.2. Relative quantification**

Such quantification measures changes in the basal state of a gene of interest versus constant gene expression that acts as a control. The difference lies in the absolute quantification that are not part of a known amount of DNA, but an endogenous housekeeping gene control or reference "housekeeping". Because the absolute amount of internal standard is unknown, can be determined only relative changes of the gene of interest with reference to the endogenous gene. Some of the most commonly used reference genes include glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, hypoxanthine guanine phosphoribosyl transferase (HPRT) and 18S ribosomal RNA.

The advantage of using mass units and normalizing the experimental design is that it is conceptually simple, but requires accurate quantitation of starting material to be used as a normalizer. There are some methods and models to determine the amount of DNA that are based on comparison of the CT values using the efficiency of PCR reaction as a correction factor. However, there is also a model that does not require the reaction efficiency of accessing a correction factor, assuming 100% efficiency in the PCR reaction in real time, both study gene as reference gene, this Method 2 is delta-delta CT (2-ΔΔCT) (Vinuesa, 2009; Aguilera et al, 2014.). Method 2-ΔΔCT expresses the ratio obtained from the relationship between the CT values of the sample and control values CT. Assay validation is made by serial dilution problem for both the endogenous gene to gene. ΔCt values (CT gen – CT endogenous) are obtained. These are plotted on the "y" axis versus the logarithm of the concentration in each of the dilutions in the "x" axis. The slope of the line should be less than or equal to 0.1 so that the method is valid [69]. Table 2 shows the most commonly used primers for the detection by different variants of PCR and also are listed the ones used to estimate the parasite load of *T. gondii* in tissues.



**Table 2.** Amplification targets, primers and probes used in the diagnosis and quantification of *T. gondii* in meat.

Although there are several methods for the diagnosis of toxoplasmosis, the present work focused on the most used by researchers working with *T. gondii* in animal tissue samples. In this aspect the molecular, especially the PCR technique and its variants, has been a major technique for diagnosing the presence and in some cases abundance of the parasite in the tissues of the various productive species that are commonly consumed tool.

The sensitivity of PCR techniques is influenced by several factors, among which are tissue type, sample handling, the process employed in the extraction and purification of nucleic acids and the type of card selected amplification. All these factors cause variation sensitivi‐ ty in these tests of about 20% to over 80% in some cases. Variations of the PCR most commonly used for the diagnosis of toxoplasmosis are conventional PCR, LAMP-PCR, nested PCR and real time PCR, considered as the most sensitive nested PCR and PCR techniques in real time, can detect the presence of nucleic acids of *T. gondii* from a single parasite in the sample.

The techniques of DNA extraction and purification significantly affect the development of PCR techniques; the presence of contaminants may inhibit the amplification process. Nowadays, there are different methodologies used for DNA extraction and also, there is a wide variety of commercial options available to perform the procedures, however, even almost all of them have been designed to obtain as much amount of DNA as possible from any specimen while minimizing manual handling and co-extraction of PCR inhibitors. Methodologies based in small-scale silica-based spin column, has proven more efficient for the recovery of pure DNA in comparison with phenol/chloroform extraction, because they have showed several disadvantages besides being unable to remove potential PCR inhibi‐ tors efficiently.

The sensitivity of PCR assays depends heavily on the number of copies of the target sequence; in the case of *T. gondii* although various cards amplification as is the case of SAG1, SAG2, SAG3 and GRA6 genes, most widely used are those which are directed to highly repetitive genes as gene B1 is repeated 35 times in the genome of *T. gondii* or repeated 529pb frac‐ tion, which is repeated 200 to 300 times in the parasite genome. Although the B1 gene is considered to be the gene of choice for the diagnosis, repeated fraction 529pb has begun to have a greater impact due to it being repeated more times increases the sensitivity of PCR testing. This trend toward the use of these cards amplification is observed in most variants of the PCR.

An important aspect for the selection of the PCR technique used in the diagnosis of toxoplas‐ mosis has been the cost involved, the techniques of real-time PCR are usually much more expensive due to the use of probes and specialized equipment for reading the results, so this type of quantitative PCR are mainly used for research.

It is important to consider that molecular methods allow us to estimate whether the presence (when we use some variants as endpoint PCR or nested PCR) or quantity (by qPCR) parasite DNA in the evaluated sample. However, these findings do not allow for determination whether the parasites are viable and capable of producing infection.

Tissues that tend to have higher parasite loads are often brain, heart and spleen, but it is feasible to detect the presence of the parasite in other tissues intended for human consump‐ tion. Loading and distribution of parasites depends on biotype and density involved in the environment, as well as the animal's age and the type of production system. Due to this high variability, it is important to determine which tissues have higher loads and biotypes which are involved in order to avoid risks of transmission to consumers of meat from different regions and emphasize the importance of giving proper management to meat products (such as cooking or freeze) to reduce the risk of acquiring infection by eating them.
