Leading commercial kits used for DNA extraction and purification from different tissues for
Abstract
Toxoplasmosis is a parasitic zoonotic disease widely distributed worldwide and is caused by the intracellular parasite Toxoplasma gondii. The definitive host of T. gondii is the domestic cat and the entire cat family, in which the sexual stages of the parasite develop. T. gondii can also infect a wide range of intermediate hosts, affecting most warm-blooded animals including humans. In humans, toxoplasmosis is usually asymptomatic in healthy individuals, but can develop lymphadenopathy and nonspecific symptomatology or even be fatal in infants with congenital toxoplasmosis and in immunocompromised patients. Transmission to humans is mainly through food, especially by eating undercooked meat or meat contaminated with tissue cysts. This has led to various public health organizations worldwide monitoring programs on T. gondii in animals intended for human consumption, especially in meat samples. One of the techniques employed in the laboratory is that based on the polymerase chain reaction and some of its variants, which have proven to be valuable tools for the detection of T. gondii in tissues for human consumption and many other types of biological samples. The development of different strategies for the molecular detection of T. gondii has led to the identification and quantification methodologies varying widely among laboratories. Therefore, this chapter reviews the main methods of extraction, purification, detection and quantification of T. gondii DNA in tissue samples from different species destined for human consumption.
Keywords
- T. gondii
- meat
- DNA quantification
- parasite load
- zoonosis
1. Introduction
Toxoplasmosis is a zoonotic disease widely distributed throughout the world and is caused by the intracellular parasite
In humans, toxoplasmosis is usually asymptomatic in healthy individuals, but can develop lymphadenopathy and nonspecific symptomatology or even be fatal in infants with congenital toxoplasmosis (CT) and in immunocompromised patients (such as people with AIDS), in persons with problems in bone marrow and recipient patients of transplanted organs) [2].
The consumption of undercooked, raw or cured meat is a major mode of transmission of
2. Occurrence of toxoplasmosis
As well in humans,
In general, the occurrence infection by
3. Clinical signs in animals and humans
3.1. Toxoplasmosis in humans
The clinical spectrum of the disease varies widely and depends primarily on the immune status of the host and
3.2. Toxoplasmosis in animals
Natural infection in non-pregnant animals usually elapses without symptoms, but primary infection during pregnancy can cause embryonic death, abortion, birth weak or clinically normal but infected animals. Globally,
4. Economic impact of toxoplasmosis
The real economic impact of toxoplasmosis is difficult to estimate, because in most immunocompetent individuals, the infection goes unnoticed or has claimed clinical presentation to other diseases [22,29]. However, it is estimated that the economic impact should be very high due to the loss of one or more days of work in mild cases, treatment and care needs, sick children, especially those with mental retardation and blindness, loss of quality lifestyle and the costs of hospitalization in severe cases and the cost of monitoring pregnant women and treatment during pregnancy who are
In United States,, despite the low incidence, the economic impact of CT is high due to the severity of the infection, associated complications, treatment and social costs. CT costs have been estimated as $ 1.26 million per case and were mainly attributed to drug costs, annual losses of productivity, special education and health care costs [38]. On the other hand, in the United States, some 3,000 babies are born every year with CT and the annual cost of treating this disease is between US$ 31 and US$ 40 million [39]. The total economic impact of CT just in the United States has been estimated as over $ 7.7 billion per year, which makes it the second most important infection to humans after foodborne salmonellosis [40]. In the United Kingdom, the annual economic impact is estimated $ 12 million [26].
In most productive animals, toxoplasmosis occurs asymptomatically; however, in animal production, toxoplasmosis is considered as an economically important disease of livestock, especially sheep and goats, where it can cause early embryonic death and resorption, fetal death and mummification, abortion, stillbirth and neonatal death [41].
Regarding economic impact of infection with
In pigs, the infection with
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 aforementioned detection methods are not possible; in these cases the techniques of molecular biology have been helpful in the diagnosis of
Currently different strategies for molecular diagnosis of
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
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
Pepsin requires acidic pH for activation; it breaks the bonds between tyrosine and phenylalanine 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
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
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 homogenate, 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 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 adsorption/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
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.
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 traditional 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
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Universal Genomic DNA Extraction Kit (TaKaRa, USA) | Absorption/desorption | Silica | 10µg (2–30 mg of tissue) | [61]. |
Easy-DNA®Kit (Invitrogen) | Organic extraction | N/A | 150µg (50 mg of tissue) 5–10 ng/µL blood |
[62]. |
High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim Germany) | Absorption/desorption | Glass fiber filter | 3–9 µg (200–300 µL blood) 25–50 mg of tissue (variable amount recovered depending the tissue tested) |
[6]. [63]. |
QIAamp DNA Mini Kit (QIAGEN, Valencia, CA) | Absorption/desorption | Silica | 4–12µg (200µL blood) 25–45µg (25mg of cardiac tissue) |
[21,47,64–68]. |
TRIzol® LS Reagent (Invitrogen, Paisley, the United Kingdom) (Fenol-Cloroformo) | Organic liquid-liquid extraction | N/A | 2–3µg (for each mg of sample) | [69]. |
Phenol-Chloroform | Organic liquid-liquid extraction | N/A | --- | [70–76] |
NucleoSpin Tissue Kit (Macherey Nagel, Czech Republic) | absorption/desorption | Silica | 20–35µg (25mg of tissue) | [77]. |
Biotin labeled capture oligonucleotides and Streptavidin labeled magnetic beads (Invitrogen) | magnetic capture | Magnetic beads |
Recovered DNA from 5–100pb | [5,47,56] |
NucliSens easy MAG system (BIOMERIEUX) | magnetic capture | Magnetic Silica |
--- | [49,78,79] |
Genomic Prep Blood DNA isolation kit (Amersham Pharmacia Biotech, the United Kingdom) |
absorption/desorption | Silica | Recovered DNA from 100–200 kb | |
DNAzol® (Life technologies) | organic extraction | N/A | 3–5µg/mg from tissue | [80]. |
Qiaquick y PCR purification kit (QIAGEN, Valencia) | absorption/desorption | Silica | 10 µg DNA recovered from 100pb–10kb | [81,82] |
DNeasy Blood & Tissue Kit (QIAGEN) | absorption/desorption | Silica | 10–30ug (25mg of tissue) | [83,84] |
6. Methods for molecular detection of T. gondii in meat for consumption
The presence of
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 "
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 complementary-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
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
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
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].
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
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
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529pb Repeat Element ( |
Toxo4 Toxo5 |
CGCTGCAGGGAGGAAGACGAAAGTTG CGCTGCAGACACAGTGCATCTCGGATT |
529pb | [62,74,75] |
Gen B1 | Tg1 Tg2 |
AAAAATGTGGGAATGAAAGAG ACGAATCAACGGAACTGTAAT |
469pb | [3,21,64] |
Gen B1 | F R |
AGCCTCTCTCTTCAAGCAGCGTA TCGGAGAGAGAAGTTCGTCGCAT |
300pb | [71,83] |
|
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Gen B1 | S1 AS1 S2 AS2 |
TGTTCTGTCCTATCGCAACG ACGGATGCAGTTCCTTTCTG TCTTCCCAGACGTGGATTTC CTCGACAATACGCTGCTTGA |
580 pb 530pb |
[49] |
Gen GRA6 | GRA6-F1x GRA6-R1 GRA6-F1 GRA6-R1x |
ATTTGTGTTTCCGAGCAGGT GCACCTTCGCTTGTGGTT TTTCCGAGCAGGTGACCT TCGCCGAAGAGTTGACATAG |
546pb 351pb |
[76] |
Gen B1 | B1F1 B1R1 B1F2 B1R2 |
TCAAGCAGCGTATTGTCGAG CCGCAGCGACTTCTATCTCT GGAACTGCATCCGTTCATGAG TCTTTAAAGCGTTCGTGGTC |
194pb | [96] |
529 pb |
REF1 RER1 REF2 RER2 |
TGACTCGGGCCCAGCTGCGT-3′ CTCCTCCCTTCGTCCAAGCCTCC-3′ AGGGACAGAAGTCGAAGGGG-3′ GCAGCCAAGCCGGAAACATC-3′ |
164pb | [93,96] |
PCR LAMP | ||||
529bp |
F3 B3 FIP (F1 c-F2) BIP (B1c-B2) |
ACGAGAGTCGGAGAGGGA TGGATTCCTCTCCTACCCCT GGATCGCATTCCGGTGTCTCTTAAGATGTTTCCGGCTTGGC GACGACGCTTTCCTCGTGGTCAAGCCTCCGACTCTGTCT |
202pb | [93, 96] |
529bp |
F3 B3 FIP BIP LF LB |
CCACAGAAGGGACAGAAGTC TCCGGTGTCTCTTTTTCCAC TCCTCACCCTCGCCTTCATCTAGGACTACAGACGCGATGC TGGTTGGGAAGCGACGAGAGTTCCAGGAAAAGCAGCCAAG TCCAAGACGGCTGGAGGAG CGGAGAGGGAGAAGATGTTTCC |
202pb | [96,97] |
Gen B1 | BIP FIP F3 B3 |
TCGCAACGGAGTTCTTCCCAGTTTTGGCCTGATATTACGACGGAC TGACGCCTTTAGCACATCTGGT TTTTGATGCTCAAAGTCGACCGC GGGAGCAAGAGTTGGGACTA CAGACAGCGAACAGAACAGA |
212 pb | [96] |
|
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Gen B1 | SYBR Green™ | BE-F: CTCCTTCGTCCGTCGTAATATC BE-R: TGGTGTACTGCGAAAATGAATC |
451pb | [47] |
SAG 1 | SYBR Green™ | F: CTGATGTCGTTCTTGCGATGTGGC R: GTGAAGTG-GTTCTCCGTCGGTGT |
128pb | [61] |
Gen B1 | SYBR Green™ | F: CGTCCGTCGTAATATCAG R: GACTTCATGGGACGATATG |
98pb | [47] |
529pb |
TaqMan™ | F: CACAGAAGGGACAGAAGT R: TCGCCTTCATCTACAGTC RE probe: (JOEe)-CTCTCCTCCAAGACGGCTGG-(TAMRAf) |
94pb | [47,63,69] |
Gen B1 | TaqMan™ | GENE_B1_TG-TX2F: CTAGTATCGTGCGGCAATGTG GENE_B1_TG-TX2R: GGCAGCGTCTCTTCCTCTTTT GENE_B1_TG-TX2M1: 6-FAM)-CCACCTCGCCTCTTGG-(NFQ-MGB) |
62 pb | [79] |
Gen B1 | TaqMan™ | BE-F: CCCCACAAGACGGCTGABE-R: TGGTGTACTGCGAAAATGAATC TaqMan probe: (6-FAM)-CATTTGCAAAACAGCGGCAGCGTCT-(DQ) |
248pb | [47] |
529pb |
TaqMan™ | TOX-9F: AGGAGAGATA TCAGGACTGT AG TOX-11R: GCGTCGTCTC GTCTAGATCG TOX-TP1: (6-FAM)-CCGGCTTGGC TGCTTTTCCT-(BHQ1) |
524pb | [5,47,56,77] |
Although there are several methods for the diagnosis of toxoplasmosis, the present work focused on the most used by researchers working with
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 sensitivity 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
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 inhibitors efficiently.
The sensitivity of PCR assays depends heavily on the number of copies of the target sequence; in the case of
An important aspect for the selection of the PCR technique used in the diagnosis of toxoplasmosis 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 consumption. 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.
8. Conclusion and recommendation
Toxoplasmosis is a zoonotic disease transmitted by common foods; the occurrence of cysts of
For the diagnosis of infection in tissue samples from naturally infected animals intended for consumption, it is desirable to use the technique Taqman qPCR probes since it is a more sensitive alternative to the same qPCR detection when used with SYBR Green dye. The format for performance this technique (qPCR with a Taqman probe) is versatile, allowing to evaluate both few samples (in a research environment for example) and in environments where it is required to test a greater number of specimens in a short time period (i.e., specialized laboratories for monitoring food safety), and increasingly, inputs for this procedure are more affordable.
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