nested pcr for the detection of taenia solium dna in stool ... · 2020-06-29 · 1 1 nested pcr...

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Nested PCR for the detection of Taenia solium DNA in stool samples 1

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Franco-Muñoz C1*, Arévalo A1, Duque-Beltran S1 3

1Grupo de Parasitología. Dirección de Investigación en Salud Pública. Instituto Nacional de 4

Salud. Bogotá D.C. Colombia 5

*Corresponding author: [email protected] 6

Avenida calle 26 No. 51-20 - Zona 6 CAN. 7

Bogotá, D.C. Colombia 8

Código postal: 111321 9

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ABSTRACT 11

The traditional parasitological method to diagnose taeniasis is the microscopic observation of 12

eggs in stool samples. However, this method does not allow differentiation between Taenia 13

saginata and Taenia solium. This aim of this study was to achieve the detection of T. solium DNA 14

by polymerase chain reaction (PCR) and to evaluate the cross-reaction with other species of the 15

genus Taenia and other intestinal parasites. DNA was extracted from adult T. solium cestodes by 16

cryolysis in liquid nitrogen and with the DNA stool extraction kit from Qiagen. The detection limit 17

of the test was evaluated by DNA dilutions in water and in stool samples. DNA was extracted from 18

proglottids of T. saginata and T. crassiceps and from stool samples containing other intestinal 19

parasites using ethanol treatment, alkaline lysis, and the DNA stool extraction kit. Nested PCR 20

was used to amplify a previously described fragment of the Tso31 gene, and the PCR products 21

were analyzed by electrophoresis in 2% agarose gels followed by staining with GelRed. The 22

nested PCR of the Tso31 gene allowed the detection of T. solium DNA in stool samples with a 23

detection limit of 20 pg of parasite DNA. PCR showed no cross-reaction with T. saginata, T. 24

crassiceps, or other intestinal parasites of public health importance in Colombia. 25

KEYWORDS: Taenia solium, taeniasis, polymerase chain reaction 26

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INTRODUCTION 28

Diseases of the taeniasis/cysticercosis complex are a public health problem in countries with 29

ecoepidemiological conditions conducive to the maintenance and transmission of these parasites, 30

such as Colombia (1). Taeniasis/cysticercosis is associated with poverty and health illiteracy in 31

communities (2). Taeniasis is caused by the ingestion of larvae (cysticerci), and two species have 32

been identified to infect humans: Taenia saginata (beef tapeworm) and Taenia solium (pork 33

tapeworm). Ingestion of T. solium eggs is the cause of neurocysticercosis, a major human health 34

problem, especially due to its main complication, epilepsy (3). T. solium infections cause 35

approximately 28,000 deaths worldwide each year, and approximately 2.7 million years of life are 36

lost due to disability (4). 37

Identifying people infected with T. solium is important to administer timely treatment and interrupt 38

the transmission cycle of the parasite. The traditional diagnostic method is the direct observation 39

of parasite eggs by conventional microscopy in stool samples. However, due to the biological 40

characteristics of the release of T. solium eggs in feces, the sensitivity of the microscopic method 41

can vary between 3.9% (5) and 52.5% (6). Microscopy does not differentiate between T. saginata 42

and T. solium since the eggs are morphologically similar. 43

Diagnosis based on the detection of T. solium DNA by polymerase chain reaction (PCR) is a very 44

useful technique with 100% specificity and 97-100% sensitivity when a fragment of the Tso31 45

gene of T. solium is amplified by nested PCR (7, 8). The objective of this study was to achieve 46

the detection of T. solium DNA by PCR in stool samples and to evaluate the cross-reaction with 47

other species of the genus Taenia and other intestinal parasites. 48

METHODS 49

Ethical aspects 50

51

This study was approved and monitored by the Ethics Committee of National Health Institute of 52

Colombia in accordance with national regulations and the Declaration of Helsinki. The patients 53

signed an informed consent statement for the use of the stool sample. The animal experiments 54

were performed following the regulations and ethical codes of the Barrier Bioterium and 55

Experimentation BSL2 of the National Institute of Health of Colombia (Declaration of Helsinki) and 56

according to the three principles of laboratory animal experimentation (reduce, refine, replace). 57



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58

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Biological material 60

In vivo maintenance of T. solium in an animal model 61

Golden hamsters were infected with viable T. solium larvae (cysticerci) as described (9). The 62

animals were infected with five cysticerci orally and then subjected to immunosuppression with 63

methylprednisolone (2 mg/animal) for 15 days. After four weeks, the hamsters were sacrificed in 64

a CO2 chamber, and the adult forms of T. solium were recovered from the intestines, which were 65

lyophilized and stored at -80 °C. All animals were female. 66

67

Stool sample from uninfected hamsters 68

69

Uninfected hamsters were sacrificed in a CO2 chamber, and stool sample was recovered from 70

the large intestine. The samples were stored in absolute ethanol at room temperature. 71

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73

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Parasitologically cheked stool samples with intestinal parasites 75

Seven samples of fecal matter preserved in formalin-glycerol stored in the Biological Bank of the 76

Parasitology Group of the National Institute of Health of Colombia were used. The samples were 77

parasitologically cheked proven to have the following parasites by conventional microscopy: eggs 78

of Taenia spp., eggs of Hymenolepis diminuta, eggs of Hymenolepis nana,eggs of Ascaris 79

lumbricoides, eggs of Trichuris trichiura, eggs of Uncinaria, cysts of Giardia, cysts of Chilomastix 80

mesnili, cysts of EntamoebaHartmanni, cysts of Entamoeba coli,, cysts of Endolimax. nana, cysts 81

of Entamoebahistolytica/Entamoeba dispar complex and Blastocystis hominis. 82

83

Biological material for cross-reaction tests 84

Biological material from related parasites isolated and stored in the Biological Bank of the 85

Parasitology Group of the National Institute of Health of Colombia was used. Samples of adult-86

stage T. crassiceps and Fasciola hepatica lyophilized and stored at -80 °C as well as proglottids 87



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of T. saginata and eggs of Strongyloides stercoralis preserved in 5% formalin-glycerol at room 88

temperature were included. 89

DNA extraction 90

Obtaining DNA from lyophilized parasites 91

Cryolysis was performed in liquid nitrogen in a porcelain flask, and the DNA was extracted with 92

the QIAamp DNA stool mini kit from Qiagen following the manufacturer's instructions. The quantity 93

and quality of the extracted DNA were evaluated with a Nanodrop 2000 spectrophotometer 94

(Thermo Scientific). 95

96

Obtaining DNA from samples preserved in formalin 97

The protocol described by Hykin et al. (2015) was followed (10). Five hundred microliters of each 98

sample was centrifuged at 16,000 × g for 5 minutes to remove formalin. Next, treatment was 99

performed with decreasing concentrations of ethanol (100% to 70%) to hydrate the DNA. The 100

samples were treated with 0.5 M EDTA for 1 hour at 55 °C to inhibit DNases and then lysed with 101

ASL buffer (Qiagen) and proteinase K for 2 hours at 55 °C. After this step, the extraction protocol 102

of the QIAamp DNA stool mini kit was continued following the manufacturer's instructions 103

(Qiagen). The quantity and quality of the extracted DNA was evaluated with a Nanodrop 2000 104

spectrophotometer (Thermo Scientific). 105

Obtaining DNA from parasites preserved in formalin-glycerol 106

Biological material (proglottids or eggs) was centrifuged at 16,000 × g for 5 minutes to remove 107

formalin. After that, treatment was performed with decreasing concentrations of ethanol (100% to 108

70%) to hydrate the DNA. DNA was extracted according to a high-temperature alkaline lysis 109

protocol described by Campos et al. (2012) (11) using 0.5-mm glass beads during lysis, followed 110

by purification using the QIAamp DNA stool mini kit according to the manufacturer's instructions. 111

Obtaining DNA from stool samples preserved in ethanol 112

A total of 200 µl of fecal matter preserved in formalin was collected and centrifuged at 16,000 × g 113

for 5 minutes. Then, 1.4 ml of ASL lysis buffer (Qiagen) was added to the precipitate, and DNA 114



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extraction was continued using the QIAamp DNA stool mini kit following the manufacturer's 115

instructions. 116

Nested PCR of the Tso31 gene 117

The first PCR was performed based on the method described by Mayta et al. in 2008 (8) with the 118

following primers that yielded a 691-bp amplified fragment of the Tso31 gene of T. solium: 119

Forward primer F1 5’-ATGACGGCGGTGCGGAATTCTG-3’ 120

Reverse primer R1 5’-TCGTGTATTTGTCGTGCGGGTCTA-3’ 121

PCR was performed with 20 ng of DNA, 1X amplification buffer (20 mM Tris-HCl, 50 mM KCl), 3 122

mM MgCl2, 200 µM of each dNTP, 0.8 µM of each primer, and 0.125 U of Platinum Taq DNA 123

polymerase (Tucan Taq) in a final volume of 20 µl. 124

The product of the first round of PCR was used as a template to perform the nested PCR as 125

described by Mayta et al. (2008) (8). The following primers were used to obtain an amplified 234-126

bp fragment: 127

Forward primer F589 5’-GGTGTCCAACTCATTATACGCTGTG-3’ 128

Reverse primer R294 5’-GCACTAATGCTAGGCGTCCAGAG-3’ 129

The nested PCR was performed with 1 µl of the first PCR amplification product, 1X amplification 130

buffer (20 mM Tris-HCl, 50 mM KCl), 2.5 mM MgCl2, 200 µM of each dNTP, 0.8 µM of each 131

primer, and 0.125 U of platinum Taq polymerase (Tucan Taq) in a final volume of 20 µl. 132

A C1000 thermal cycler (Bio-Rad) was used for PCR with the following thermal profile. First PCR: 133

95 °C for 3 minutes followed by 25 amplification cycles of 95 °C for 30 seconds, 55 °C for 30 134

seconds, and 72 °C for 1 minute. Second PCR: 95 °C for 3 minutes followed by 40 amplification 135

cycles of 95 °C for 30 seconds, 50 °C for 30 seconds, and 72 °C for 1 minute. 136

The PCR products were subjected to agarose gel electrophoresis to visualize the amplified 137

fragments. Five microliters of the amplified product was loaded onto a 2% agarose gel in TBE 138

buffer solution (Tris, boric acid, EDTA) and 4 µl of GelRED. Electrophoresis was performed at 80 139

V for 1 hour, and the gel was visualized using the Gel Doc imager and Image Lab software (Bio-140

Rad). 141



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Limit of detection of nested PCR 142

Ten-fold dilutions were performed with T. solium DNA starting from a concentration of 20 ng/µl 143

and going to 20 fg/µl. The diluted DNA was used as a template to perform the nested PCR of the 144

Tso31 gene as described above. 145

Performance of the PCR in samples 146

A total of 200 µl of uninfected hamster fecal matter preserved in absolute ethanol was mixed with 147

T. solium DNA in different amounts between 2 µg and 20 pg. Then total DNA extraction was 148

performed as described above, and the Tso31 gene of T. solium was amplified to evaluate the 149

limit of detection of the PCR in a sample matrix similar to that expected for clinical samples. 150

151

RESULTS 152

It was possible to reproduce the PCR conditions reported by Mayta et al. (2008) using as template 153

the DNA extracted from the adult form of T. solium recovered from intestines of infected hamsters 154

in the laboratory. The amplified fragments of the Tso31 gene in the first and second PCR rounds 155

matched the expected size as reported in the literature and predicted by in silico analysis (Fig. 156

1A). Nested PCR can specifically amplify the DNA of T. solium without cross-reacting with other 157

related species of the genus Taenia, such as T. saginata and T. crassiceps (Fig. 1B), or with other 158

intestinal parasites, such as Strongyloides stercoralis and Fasciola hepatica (Fig. 1C). 159

The limit of detection of PCR in stool samples containing T. solium DNA was a proportion of 20 160

ng of DNA in 200 µl of stool sample (Fig. 2A). However, considering that the DNA was added 161

before extraction, the PCR performance may have been influenced by the presence of DNases 162

in the samples, PCR inhibitors, and the extraction yield. To evaluate the PCR performance under 163

the best possible conditions, serial dilutions of T. solium DNA were performed in water, and 164

amplification of the Tso31 gene fragment was recorded with up to 20 pg of DNA (Fig. 2B), 165

suggesting that the PCR limit of detection varied between 20 ng and 20 pg of DNA, depending on 166

the sample characteristics. 167

The results of the nested PCR of the Tso31 gene in DNA extracted from human stool samples 168

with microscopic verification of Taenia spp. and other intestinal parasites are shown in Fig. 3. All 169

samples were negative, suggesting that the nested PCR test did not cross-react with any of the 170



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parasites present in the different samples (Fig. 3A). In the clinical samples with microscopic 171

verification of the presence of Taenia eggs, no amplification of the Tso31 gene was found, 172

suggesting that in these samples, the infecting species could be T. saginata. To rule out that the 173

negative results were due to the presence of PCR inhibitors in the extracted DNA, considering 174

that these were stool samples stored for more than 5 years in formalin-glycerol, the experiment 175

was repeated with the addition of 20 ng of DNA of T. solium in 200 µl of the sample before 176

performing total DNA extraction. In these spiked samples, it was possible to amplify the Tso31 177

gene fragment, suggesting that the negative results were not due to PCR inhibitors (Fig. 3B). This 178

test was performed on all negative samples (results not shown). y no se detectó ADN de T. solium 179

DISCUSSION 180

Nested PCR of a fragment of the Tso31 gene can specifically detect DNA from T. solium in stool 181

samples without showing cross-reaction with other species of the genus Taenia or other intestinal 182

parasites of public health importance in Colombia. Therefore, this is an alternative for the 183

detection of the parasite that can be considered in laboratories that have the necessary 184

infrastructure to perform molecular biology tests. 185

The PCR detection limit we found is similar to that reported in a study that amplified fragments of 186

the Cox1, Nad5, and 12S genes in a general PCR protocol for the detection of DNA of parasites 187

of the class Cestoda. The authors found that the PCR detection limit could vary by several orders 188

of magnitude, depending on the type of sample and the molecular target chosen, similar to what 189

was found in the present study (12). 190

No PCR cross-reaction was found with DNA from other intestinal parasites or with DNA extracted 191

from adult forms of T. saginata and T. crassiceps. The results showed the amplification of a single 192

234-bp fragment only in the samples with T. solium DNA. However, Vargas-Calla et al. (2019) 193

evaluated whether the Tso31 gene was conserved in other Taenia species and found that PCR 194

of it was negative in DNA from T. omisa, T. hydatigena, T. pisiformis, and T. taeniaeformis but 195

was positive in T. multiceps; the authors suggested that the Tso31 gene sequence is not exclusive 196

to T. solium (13). Considering the above, it is important to continue evaluating the possible cross-197

reactions of this test with other, related parasites. 198

For the PCR technique, 100% specificity and 97-100% sensitivity are reported (8), whereas the 199

sensitivity of the microscopic method can vary between 3.9% (5) and 52.5% (6). In addition to the 200

high sensitivity and specificity, the PCR method offers the advantage of differentiating between 201



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T. saginata and T. solium, which favors the identification of the infecting species, as in the case 202

of the samples included in the present study, which, despite microscopic verification of the 203

presence of Taenia eggs, were negative for T. solium. 204

Immunodiagnosis based on the detection of T. solium antigens in stool samples is another method 205

that has been suggested to overcome the limitations of microscopic diagnosis. For this method, 206

sensitivity rates between 96.4% and 98% and specificity rates between 99.2% and 100% are 207

reported (5, 14). However, the level of species-specific specificity reported for these tests is only 208

reached after the recovery of secretion/excretion antigens obtained by the infection of animal 209

models with larvae of different Taenia species and the recovery of the parasite in its adult stage 210

(14). This can be avoided with the nested PCR evaluated in this study, but this technique can only 211

be applied in laboratories equipped with the necessary infrastructure. 212

The possibility to bring parasite DNA-based detection tests, such as PCR, to the patient point of 213

care can be achieved through the development of loop-mediated isothermal amplification 214

techniques. For this type of test, a sensitivity of 88.4% is reported for the diagnosis of T. solium 215

infections (15). However, this method requires further development to achieve better operational 216

characteristics (16). 217

One of the challenges of this study was the extraction of DNA from various types of samples, 218

especially those preserved in formalin. Formalin-fixed biological samples stored in biological 219

banks are of great importance for research based on genomic resources. However, the quality of 220

the DNA is very poor due to the formalin-induced fragmentation and cross-linking of DNA with 221

proteins (10). It is difficult to predict the quality of the extracted DNA because the levels of 222

degradation and cross-linking with proteins may depend on factors such as pH, temperature, the 223

ionic strength of the buffer, and the storage time of the samples under these conditions (11). 224

Nested PCR of the Tso31 gene is a useful method for detecting T. solium DNA in stool samples 225

and could be adopted by programs to control and/or eliminate the taeniasis/cysticercosis complex. 226

Funding: This study was funded by the National Institute of Health of Colombia. 227

Acknowledgments: The authors thank María Teresa Herrera and Lyda Muñoz for their support 228

in the laboratory of the Parasitology Group of the Public Health Research Directorate of the 229

National Institute of Health of Colombia. 230

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REFERENCES 232

1. Flórez Sánchez AC, Pastrán SM, Vargas NS, Beltrán M, Enriquez Y, Peña AP, et al. 233

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en México Limusa-Noriega, CONACYT. 1989;266. 237

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management strategies in endemic countries. Risk Manag Health Policy. 2017;10:107-16. 239

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5. Allan JC, Velasquez-Tohom M, Torres-Alvarez R, Yurrita P, Garcia-Noval J. Field trial of 243

the coproantigen-based diagnosis of Taenia solium taeniasis by enzyme-linked immunosorbent 244

assay. AJTMH. 1996;54(4):352-6. 245

6. Praet N, Verweij JJ, Mwape KE, Phiri IK, Muma JB, Zulu G, et al. B ayesian modelling to 246

estimate the test characteristics of coprology, coproantigen ELISA and a novel real‐time PCR for 247

the diagnosis of taeniasis. Trop Med Int Health. 2013;18(5):608-14. 248

7. Jimenez JA, Rodriguez S, Moyano LM, Castillo Y, García HH, Peru CWGi. Differentiating 249

Taenia eggs found in human stools: does Ziehl‐Neelsen staining help? Trop Med Int Health. 250

2010;15(9):1077-81. 251

8. Mayta H, Gilman RH, Prendergast E, Castillo JP, Tinoco YO, Garcia HH, et al. Nested 252

PCR for specific diagnosis of Taenia solium taeniasis. J Clin Microbiol. 2008;46(1):286-9. 253

9. Verster AJ. The golden hamster as a definitive host of Taenia solium and Taenia saginata. 254

1974. 255

10. Hykin SM, Bi K, McGuire JA. Fixing Formalin: A Method to Recover Genomic-Scale DNA 256

Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing. 257

PLoS One. 2015;10(10):e0141579. 258

11. Campos PF, Gilbert TM. DNA extraction from formalin-fixed material. Methods Mol Biol. 259

2012;840:81-5. 260

12. Roelfsema JH, Nozari N, Pinelli E, Kortbeek LM. Novel PCRs for differential diagnosis of 261

cestodes. Exp Pparasitol. 2016;161:20-6. 262



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13. Vargas-Calla A, Gomez-Puerta LA, Lopez MT, Garcia HH, Gonzalez AE, Peru CWGi. 263

Molecular characterization of the Taenia solium Tso31 antigen and homologous of other Taenia 264

species from Peru. Parasitol Rres. 2019;118(4):1307-9. 265

14. Guezala M-C, Rodriguez S, Zamora H, Garcia HH, Gonzalez AE, Tembo A, et al. 266

Development of a species-specific coproantigen ELISA for human Taenia solium taeniasis. 267

AJTMH. 2009;81(3):433-7. 268

15. Nkouawa A, Sako Y, Li T, Chen X, Wandra T, Swastika IK, et al. Evaluation of a loop-269

mediated isothermal amplification method using fecal specimens for differential detection of 270

Taenia species from humans. J C Microbiol. 2010;48(9):3350-2. 271

16. Sako Y, Nkouawa A, Yanagida T, Ito A. Loop-mediated isothermal amplification method 272

for a differential identification of human Taenia tapeworms. Nucleic Acid Detection: Springer; 273

2013. p. 109-20. 274

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Fig. 1. Nested PCR specifically amplifies the DNA of T. solium and not that of T. saginata, 298

T. crassiceps, or other intestinal parasites. A. Amplification of Tso31 gene fragments using 299

the conditions described by Mayta et al. and T. solium DNA as template. B. Amplification of the 300

Tso31 gene by nested PCR using DNA from different species of the genus Taenia recovered from 301

humans and mice as templates. C. Amplification of the Tso31 gene by nested PCR using DNA 302

from Strongyloides stercoralis and Fasciola hepatica isolated from humans. All experiments were 303

performed in duplicate. Positive control: DNA extracted from T. solium in adult form recovered 304

from hamster. Negative control: reaction mixture without template. 305

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Fig. 2 Tso31 nested PCR has a detection limit of 20 pg of DNA in water and 20 ng in 200 µl 313

of fecal matter. A. Amplification of a fragment of the Tso31 gene by nested PCR in samples of 314

uninfected hamster fecal matter preserved in absolute ethanol with T. solium DNA in different 315

amounts between 2 µg and 20 pg. All experiments were performed in duplicate. Positive control: 316

DNA extracted from T. solium in adult form recovered from hamster. Negative control: reaction 317

mixture without template. B. Amplification of a fragment of the Tso31 gene by nested PCR in 318

different dilutions of T. solium DNA in water between 20 ng and 20 fg. 319

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Fig. 3 The PCR is negative in human fecal samples with parasitological verification of 326

different intestinal parasites. A. The results of the nested PCR of the Tso31 gene on DNA 327

extracted from samples of human fecal material preserved in formalin-glycerol with parasitological 328

verification by microscopy. Sample 39: Taenia spp. Entamoeba coli cysts; Sample 123: eggs of 329

Hymenolepis diminuta, E. coli cysts, Giardia cysts, Ascaris eggs, Trichuris trichiura; Sample 454: 330

Uncinaria cysts, B. hominis, Giardia cysts; Sample 458: Ascaris eggs, Trichuris trichiura, E. coli 331

cysts, E. hartmani cyst, Chilomastix mesnili; Sample 460: Hymenolepis nana, E. coli cysts, E. 332

nana, Giardia cysts, E. histolytica complex; Sample 461: Ascaris eggs; Sample 416: Uncinaria 333

eggs, Ascaris eggs, Trichuris trichiura, E. coli cysts, Giardia cysts. All experiments were 334

performed in duplicate. Positive control: DNA extracted from T. solium in adult form recovered 335

from hamster. Negative control: reaction mixture without template. B. Evaluation of the presence 336

of PCR inhibitors in the DNA extracted from human fecal material samples and preserved in 337

formaldehyde-glycerol; 20 ng of T. solium DNA in 200 µl of sample before performing total DNA 338

extraction. 339



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