Entamoeba histolytica and Entamoeba dispar real-time PCR (2,3)
Principle
The E. histolytica/E. dispar specific primers, forward primer Ehd-239F, and
reverse primer Ehd-88R amplify a 172-bp fragment inside the SSU rRNA gene. The
XS-probe* histolytica-96T is used to detect E. histolytica specific amplification, and
XS- probe* dispar-96T to detect E. dispar specific amplification. PhHV-1-specific
primers and probe set consists of forward primer PhHV-267s, reverse primer PhHV337as and the double-labelled probe PhHV-305tq.
*Minor Groove Binding probes that were used in the original description can also be
used but might show some spectral overlap of the signals using some real-time PCR
systems.
Method
Amplification reactions are performed in white PCR plates, in a volume of 25
µl with PCR buffer (HotStarTaq mastermix, Qiagen), 5 mM MgCl2, 12.5 pmol of each
E. histolytica/E. dispar specific primer, 7.5 pmol of each PhHV-1 specific primer, 1.25
pmol of E. histolytica specific XS-probe, 1.25 pmol of E. dispar specific XS-probe,
1.25 pmol of PhHV-1 specific double-labelled probe and 5 µl of the DNA sample.
Amplification consists of 15 min at 95ºC followed by 50 cycles of 15 s at 95ºC, 30 s at
60ºC, and 30 s at 72ºC. Negative and positive control samples are included in each
amplification run. All primers and detection probes are given in table 2.
Table 1
Worksheet for 100 samples
Components:
Concentration:
H20
MgCl2
BSA
Primer Ehd - F
Primer Ehd - R
Probe histo - MGB - VIC
Probe dispar - MGB - FAM
Primer PHHV - 267S
Primer PHHV - 337AS
Probe PHHV - 305TQ - Cy5
HotStar Taq Master Mix
Total
Add 5 µl DNA to the mix
Table 2
1 Sample:
25 mM
5 mg/ml
25 µM
25 µM
10 µM
10 µM
25 µM
25 µM
10 µM
Mix:
2,575
3,50
0,50
0,125
0,125
0,125
0,125
0,15
0,15
0,125
12,50
20,00
257,50
350,00
50,00
12,50
12,50
12,50
12,50
15,00
15,00
12,50
1250,00
2000,00
Oligonucleotide primers and detection probes for real-time PCR for
the simultaneous detection of Entamoeba histolytica, Entamoeba dispar, and Phocin
Herpes Virus 1 as an internal control
Target organism
Genbank accession
Oligonucleotide name
Oligonucleotide sequence
no. and literature
reference of target
sequence
Entamoeba histolytica/
Entamoeba dispar
Ehd-239F
5’-ATTGTCGTGGCATCCTAACTCA-3’
X64142/Z49256, (3)
Ehd-88R
5’-GCGGACGGCTCATTATAACA-3’
X64142/Z49256, (3)
Ehistoltycia-96_MGB
VIC 5’-TCATTGAATGAATTGGCCATTT-3’ NFQ
X64142, (3)
Edispar-96_MGB
FAM 5'- TTACTTACATAAATTGGCCACTTTG-3' NFQ
Z49256, (3)
Phocin Herpes Virus
PhHV-267s
5’-GGGCGAATCACAGATTGAATC -3’
(1)
PhHV-337as
5’-GCGGTTCCAAACGTACCAA -3’
(1)
PhHV-305tq
Cy5 5’-TTTTTATGTGTCCGCCACCATCTGGATC-3’-BHQ2
(1)
Reference List
1. Niesters, H. G. 2002. Clinical virology in real time. J.Clin.Virol. 25 Suppl 3:S3-12.
2. Qvarnstrom, Y., C. James, M. Xayavong, B. P. Holloway, G. S. Visvesvara, R.
Sriram, and A. J. Da Silva. 2005. Comparison of real-time PCR protocols for
differential laboratory diagnosis of amebiasis. Journal of Clinical Microbiology 43:54915497.
3. Verweij, J. J., F. Oostvogel, E.A.T. Brienen, A. Nang-Neifubah, J. Ziem, and A.M.
Polderman. 2003 Prevalence of Entamoeba histolytica and Entamoeba dispar in
northern Ghana. Tropical Medicine and International Health 8:1153-1156.
July 2010
Department of Parasitology – Section Epidemiology and Diagnostics
LUMC – Leiden
The Netherlands
Website: http://www.lumc.nl/con/1040/81028091348221/811071047002556/