Detection of Toxoplasma gondii by Quantitative PCR

DETECTION OF TOXOPLASMA GONDII BY PCR AND

QUANTITATIVE PCR WITH HIGH SPECIFICTY AND
LOWER LIMIT OF DETECTION
Chavaphat Suviriyapaisal1,2, Darawan Wanachiwanawin1 and
Suvit Limawongpranee1,2

Department of Parasitology, 2Graduate Program in Immuology, Department of

1

Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok,

Thailand

Abstract. Toxoplasmosis is one of the important zoonotic protozoan diseases,

which can occur in both animals and humans. It is a leading cause of abortion in
newborn, blindness and brain abscesses in immunodeficient patients. Traditional
molecular assays often are difficult to perform, especially for early diagnosis of
Toxoplasma gondii infection. The objective of this study was to develop and evaluate
PCR and quantitative (q)PCR assay with a lower limit of detection and specificity
targeting multi-copy B1 of T. gondii. The detection limit of T. gondii DNA by both
PCR and qPCR was 10 ag, equivalent to 0.01 tachyzoite/µl. The specificity of qPCR
was confirmed using genomic DNA from Entamoeba histolytica, Escherichia coli,
Neospora caninum, Cryptosporidium spp, Cryptococcus neoformans, Giardia duodena-
lis, Klebsiella spp, Mycobacterium tuberculosis, cat, dog, swine, cow, and humans.
Keywords: Toxoplasma gondii, detection limit, PCR, quantitative PCR

INTRODUCTION Gangneux and Darde, 2012) . In animal,

Toxoplasma gondii is an obligate in-
tracellular protozoan parasite, which
can infect most warm blooded animals
(Blader and Saeij, 2009). The parasite
does not make any serious indisposition
for healthy humans, but on the other
hand, it can cause blindness and mental
retardation in congenital infection and
severe disease in those with suppressed
immunity, eg, patient with HIV-AIDS and
organ transplantation recipients (Robert-
Correspondence: Suvit Limawongpranee, De-
partment of Parasitology, Faculty of Medicine
Siriraj Hospital, Mahidol University, Bangkok
10700, Thailand.
Tel: +66 (0) 81 6968112
E-mail: suvit.lima@gmail.com
severity of infection has been found in
many species, such as embryonic death,
fetal death, mummification, abortion, still-
birth, and neonatal death (Dubey, 2009).
In northern Thailand, prevalence of
toxoplasmosis is 30.6% in uveitis patients
(Sirirungsi et al, 2009). Wanachiwanawin
et al (2001) found it is 53.7% in HIV-se-
ropositive and 5.3% in non-HIV infected
women who attended the antenatal-care
clinic, Siriraj Hospital, Bangkok. In addi-
tion, Sukthana et al (2000) reported 72.6%
T. gondii seropositive in patients within a
year after receiving kidney transplanta-
tion during which high dose of immuno-
suppressive drugs were used, and 43.2%
of HIV positive with T. gondii antibody
positivity have signs and symptoms of

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 Southeast Asian J Trop Med Public Health
acute toxoplasmosis involving the eye
and/or central nervous system.
Serological tests used for detecting
IgM and IgG, such as enzyme-linked
immnuno-absorbant assay (ELISA) (Abu-
Madi et al, 2008; El-Gozamy et al, 2009),
latex agglutination test (Dawoud et al,
2009; El-Gozamy et al, 2009) and indirect
immunofluorescence assay (IFA) (Da-
woud et al, 2009) are primary methods for
routine diagnosis of toxoplasmosis. How-
ever, distant past and recent infections and
in cases of immunocompromised patients
may produce variable serological results
(Tekkesin et al, 2011).
Therefore, attempts have been made
to develop more efficient and reliable
methods based on PCR and quantitative
polymerase chain reaction (q)PCR plat-
forms (Bastien et al, 2008). In this study,
the target gene used to detected T. gondii
was B1, which has 35 copies (Grigg and
Boothroyd, 2001). Primers were designed
to amplify a conserved sequence present
in B1 gene among various strains and
isolates of T. gondii (Fallahi et al, 2014).
Specificity of the assay using an array of
genomes of fungal and bacterial species
as well as hosts. Linearity and precision
of the qPCR assay were evaluated.
MATERIALS AND METHODS
Cell culture and preparation of T. gondii
African green monkey (Vero) cells ob-
tained from Department of Protozoology,
Faculty of Tropical Medicine, Mahidol
University were used as cell line. Vero
cells were grown in modified Eagle me-
dium (Thermo Fisher Scientific, Waltham,
MA) supplemented with L-glutamine,
fungizone, penicillin-streptomycin, and
10% fetal calf serum (FCS) (Thermo Fisher
Scientific, Waltham, MA) at 37ºC under
750
5% CO 2 atmosphere until there was a
complete confluency. Then cells were
transferred to modified Eagle medium
containing 2% FCS and incubated as de-
scribed above for 24 hours. T. gondii (1x106
cells) was added to the cell culture, which
was then incubated as described above
for 48 hours. Vero cells and T. gondii were
harvested and centrifuged at 500g for 1-2
minutes to sediment dead host cells. The
supernatant was centrifuged at 100g for
10 minutes to sediment T. gondii and used
for DNA extraction.
DNA extraction
DNA was extracted from T. gondii
and other microorganisms: Entamoeba
histolytica, Escherichia coli, Neospora cani-
num, Cryptosporidium spp, Cryptococcus
neoformans, Giardia duodenalis, Klebsiella
spp, and Mycobacterium tuberculosis, and
from feline, canine, swine, bovine, and
human cells using NucleoSpin® Tissue ex-
traction kit (MACHEREY-NAGEL, Düren,
Germany). DNA concentration was de-
termined spectrophotometrically using
NanoDrop ® ND-1000 (Thermo Fisher
Scientific). Extracted genomic DNA was
stored at -20oC until used.
Optimization of PCR and qPCR assays
PCR. Primers, designed using http://
www.ncbi.nlm.nih.gov/pubmed, were F
5′-AAATACAGGTGAAATGTACCTC-
CAGAAAAG-3′ (Tm 63.4oC) and R 5′-CTC-
TACAAAATCCAACTCCTGGTGTACT-3′
(Tm 61.8oC), generating an amplicon of
124 bp. The optimal PCR assay in a 20-µl
volume contained 1.5 mM MgCl2 (2 µl),
200 mM each dNTP, 2 µl of PCR buffer
(Fermentas, St Leon-Rot, Germany), 2 µl of
4 mM each primers, 2 µl of DNA and 2.5 U
Taq DNA pol (Fermentas). Thermocycling
was performed in a Mycycler thermal cy-
cler (Bio-Rad, Hercules, CA) as follows: 5
minutes at 95ºC; followed by 45 cycles of
Vol 48 No. 4 July 2017
 Detection of Toxoplasma gondii by Quantitative PCR

500
400
300
200

100

Fig 1–Electrophorogram of Toxoplasma gondii tachyzoite 124-bp amplicon. DNA extracted from T.
gondii tachyzoites grown in cell culture was amplified using primers targeting multi-copy Bt.
Lane M, DNA size markers; lane Neg, negative control (distilled water). Number refers to
number of tachyzoites based on 10-fold dilution of extracted DNA.

60 seconds at 95ºC, 45 seconds at 58ºC, and Evaluation of sensitivity

60 seconds at 72ºC; with a final step of 5
minutes at 72ºC. Amplicons were analyzed
by 1.5% agarose gel-electrophoresis for 1
hour at 90 V, 240 mA in 1X TBE buffer (0.04
M Tris-borate-HCl), (JT Baker, Mexico City,
Maxico). Gels were stained with ethidium
bromide (Sigma, St Louis, MO) (0.5 µg/
ml) and visualized under UV light (Gel
Doc, Bio-Rad, Hercules, CA). All reactions
were conducted at least in duplicate and
negative controls (distilled water) also
were included.
QPCR. QPCR was only performed (in Ro-
tor Gene 6000, Corbett Life Science, Syd-
ney, Australia) when T. gondii DNA was
detected by PCR. Assay was conducted
in a 20-μl reaction containing 10 μl of SSo-
FastTM fluorescent dye (Bio-Rad), 10 mmol
of each primer and 2 µl of DNA template
(Department of Protozoology, Faculty of
Tropical Medicine , Mahidol University).
Thermocycling program consisted of 45
cycles of 10 seconds at 98°C, 20 seconds
at 63°C and 20 seconds at 72°C. Then a
melting curve was generated by heating
the solution from 75oC to 95oC using a
heating rate of 1oC/second with continu-
ous monitoring of fluorescence.
PCR. Serial 10-fold dilutions of T. gondii
DNA were carried out, ranging from 10 pg
to 10 ag, equivalent to 104 to 10-2 tachyzoites.
Each experiment was conducted in trip-
licate
QPCR. A standard concentrate curve was
generated using 10-fold serial dilution of
T. gondii DNA ranging from 100 fg/µl to 10
ag/µl, equivalent to 102 to 10-2 tachyzoites,
versus threshold cycle (CT). Each experi-
ment was performed in triplicate.
Evaluation of specificity of PCR and qPCR
Specificity of the PCR and qPCR
assays was conducted by testing DNA
prepared from samples described above.
Ethical statement
The study was approved by the Hu-
man Research Ethics Committee, Faculty
of Medicine Siriraj Hospital, Mahidol
University [313/2554(EC3)].
RESULTS
Performance of PCR assay in T. gondii
detection
The lower limit detection of T. gondii
as tested by PCR based on ethidium bro-

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 Southeast Asian J Trop Med Public Health

100

10

0.1

0.01

5 10 15 20 25 30 35 40
Cycle
Fig 2–Fluorescence of Toxoplasma gondii tachyzoite 124-bp. DNA extracted from T. gondii tachyzoites
grown in cell culture was amplified by quantitative PCR using primers targeting multi-copy
Bt. Number refers to number of tachyzoites based on 10-fold dilution of extracted DNA.

7
6.5
100
6
5.5 Dimer
10
5
4.5
4 1
dF/dT

3.5
3
0.1
2.5
2
1.5 0.01
1
.5
0

Fig 3–Melting curve of amplicons generated in Fig 2.

mide-stained 124 bp bands in agarose gel
was equivalent to 10-2 tachyzoite (Fig 1).
Performance of qPCR in T. gondii DNA
detection
Based on measurable CT value the
limit of detection was 10-2 tachyzoite/μl
(Fig 2). Specificity of the designed primer
pair targeting T. gondii B1 was manifested
by a single melting curve with a Tm of
83.3-85.5°C with minimal presence of
primer-dimer amplicon (Fig 3). The stan-
dard curve was linear, with regression

752 Vol 48 No. 4 July 2017

 Detection of Toxoplasma gondii by Quantitative PCR

31

30

29

28

27

26

25
CT

24

23

22

21

20

19

18

Concentration
Fig 4–Plot of threshold cycle (CT) versus log tachyzoite number obtained from Fig 2.

(R) of 0.99660 and R2 of 0.99321 (slope = in which a segment of the genome of T.

3.525) (Fig 4). This standard curve had an
efficiency of 0.92, typically required for
optimal results.
Evaluation of specificity
The specificity of qPCR was evaluated
using DNA from Entamoeba histolytica,
Klebsiella spp, Mycobacterium tuberculosis,
Cryptococcus neoformans, Escherichia coli,
Cryptosporidium spp, Giardia lamblia, Neos-
pora caninum, dog, cat, swine, cow, and
humans. There were no evidences that the
T. gondii B1-specific primers amplified any
of the above DNA samples as evidenced
by the absence of any fluorescence signal
above threshold value even after 40 cycles
of amplification (data not shown).
DISCUSSION
For implementation of a test pro-
cedure for routine use, it is essential to
establish a validation methodology. PCR
gondii is detectable has an advantage of
sensitivity and specificity over other non-
molecular techniques (Jones et al, 2000).
Obtaining immediate results is also an-
other advantage (James et al, 1996). PCR
has been used to detect T. gondii in many
types of tissues and clinical specimens
(Sukthana et al, 2003; Wiengcharoen et al,
2004; Mahittikorn et al, 2005). How-
ever, there are reports of clinical samples
containing compounds inhibiting PCR
(Chabbert et al, 2004) . The clinical sample
could noticeably reform by further in vivo
as it is used in normal state but not in an
inflammation condition.
We discovered after using qPRC that
there was an association among the high
DNA loads which interprets as a high
copy figure of DNA.The limit of both
PCR and qPCR detection reached 10 ag or
0.01 tachyzoite/μl, which should be sensi-
tive enough even to detect asymptomatic

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patients. This suggests that the test can be
used without any further modification,
even for DNA extracted from clinical
samples. It should be mentioned that
qPCR is not always more sensitive than
conventional PCR (Bastien et al, 2008). The
limit of detection in previous studies is 0.1
tachyzoite/µl (Montoya et al, 2010) and 2
tachyzoites/sample (Jalal et al, 2004). Fal-
lani et al (2014), using duplicate RE-nested
PCR, reported a detection limit of 640 fg of
T. gondii DNA, whereas that of B1-nested
PCR is 5.12 pg.
In conclusion, our study demon-
strates that a lower limit of T. gondii de-
tection with PCR and qPCR techniques
was possible using primers specifically
targeting T. gondii multi-copy B1. Besides
their diagnostic application in human
infection, the PCR assays developed in
this report should be extended to studies
of Toxoplasma gondii infection in animals
to determine the extent of this zoonotic
parasite in Thailand.
REFERENCE
Abu-Madi MA, Al-Molawi N, Behnke JM.
Seroprevalence and epidemiological
correlates of Toxoplasma gondii infections
among patients referred for hospital-based
serological testing in Doha, Qatar. Parasit
Vectors 2008; 1: 39.
Bastien P, Procop GW, Reischl U. Quantitative
real-time PCR is not more sensitive than
“conventional” PCR. J Clin Microbiol 2008;
46: 1897-900.
Blader IJ, Saeij JP. Communication between
Toxoplasma gondii and its host: impact on
parasite growth, development, immune
evasion, and virulence. APMIS 2009; 117:
458-76.
Chabbert E, Lachaud L, Crobu L, Bastien P.
Comparison of two widely used PCR
primer systems for detection of toxoplas-
ma in amniotic fluid, blood, and tissues. J
754
Clin Microbiol 2004; 42: 1719-22.
Dawoud HA, Ageely HM, El Shake AH, Heiba
AA. Latex agglutination and indirect im-
munofluorescence tests in the diagnosis of
Toxoplasma gondii in Saudi Arabia. J Egypt
Soc Parasitol 2009; 39: 1-9.
Dubey JP. Toxoplasmosis in sheep--the last 20
years. Vet Parasitol 2009; 163: 1-14.
El-Gozamy BR, Mohamed SA, Mansour HA.
Toxoplasmosis among pregnant women
in Qualyobia Governorate, Egypt. J Egypt
Soc Parasitol 2009; 39: 389-401.
Fallahi S, Kazemi B, Seyyed Tabaei SJ, et al.
Comparison of the RE and B1 gene for
detection of Toxoplasma gondii infection in
children with cancer. Parasitology Int 2014;
63: 37-41.
Grigg ME, Boothroyd JC. Rapid identification
of virulent type I strains of the protozoan
pathogen Toxoplasma gondii by PCR-re-
striction fragment length polymorphism
analysis at the B1 gene. J Clin Microbiol
2001; 39: 398-400.
Jalal S, Nord CE, Lappalainen M, Evengard, B.
Rapid and sensitive diagnosis of Toxoplas-
ma gondii infections by PCR. Clin Microbiol
Infect 2004; 10: 937-9.
James GS, Sintchenko VG, Dickeson DJ, Gilbert
GL. Comparison of cell culture, mouse
inoculation, and PCR for detection of
Toxoplasma gondii: effects of storage condi-
tions on sensitivity. J Clin Microbiol 1996;
34: 1572-5.
Jones CD, Okhravi N, Adamson P, Tasker S,
Lightman S. Comparison of PCR detec-
tion methods for B1, P30, and 18S rDNA
genes of T. gondii in aqueous humor. Invest
Ophthalmol Vis Sci 2000; 41: 634-44.
Mahittikorn A, Wickert H, Sukthana Y. Com-
parison of five DNA extraction methods
and optimization of a b1 gene nested PCR
(nPCR) for detection of Toxoplasma gondii
tissue cyst in mouse brain. Southeast Asian
J Trop Med Public Health 2005; 36: 1377-82.
Montoya A, Miro G, Blanco MA, Fuentes I.
Comparison of nested PCR and real-time
Vol 48 No. 4 July 2017
 Detection of Toxoplasma gondii
PCR for the detection of Toxoplasma gon-
dii in biological samples from naturally
infected cats. Res Vet Sci 2010; 89: 212-3.
Robert-Gangneux F, Darde ML. Epidemiology
of and diagnostic strategies for toxoplas-
mosis. Clin Microbiol Rev 2012; 25: 264-96.
Sirirungsi W, Pathanapitoon K, Kongyai N, et
al. Infectious uveitis in Thailand: serologic
analyses and clinical features. Ocul Immu-
nol Inflamm 2009; 17: 17-22.
Sukthana Y, Chintana T, Lekkla A. Toxoplasma
gondii antibody in HIV-infected persons. J
Med Assoc Thai 2000; 83: 681-684.
Sukthana Y, Waree P, Pongponratn E, Chaisri
U, Riganti M. Pathologic study of acute
toxoplasmosis in experimental animals.
Southeast Asian J Trop Med Public Health
Vol 48 No. 4 July 2017
by Quantitative PCR
2003; 34: 16-21.
Tekkesin N, Keskin K, Kilinc C, Orgen N, Molo
K. Detection of immunoglobiulin G anti-
bodies to Toxoplasma gondii: evaluation of
two commercial immunoassay systems.
J Microbiol Immunol Infect 2011; 44: 21-6.
Wanachiwanawin D, Sutthent R, Chokephaib-
ulkit K, Mahakittikun V, Ongrotchanakun
J, Monkong N. Toxoplasma gondii antibod-
ies in HIV and non-HIV infected Thai
pregnant women. Asian Pac J Allergy Im-
munol 2001; 19: 291-3.
Wiengcharoen JT, Chiabchalard R, Sukthana
Y. PCR technique for detecting Toxoplasma
gondii in animal amniotic fluid. Southeast
Asian J Trop Med Public Health 2004; 35:
792-5.
755