RAPID DETECTION OF HUMAN TORQUE TENO VIRUSES
USING HIGH-RESOLUTION MELTING ANALYSIS
Spandole S1*, Cimponeriu D1, Toma M1, Radu I1, Ion DA2
*Corresponding Author: Ms. Sonia Spandole (Ph.D. Student), Department of Genetics, University of Bucharest,
Intrarea Portocalelor Street, No 1-3, 060101, Bucharest, Romania; Tel.: 004-0764-824-281, Fax: 004-0213-181-
565; E-mail: sonia.spandole@gmail.com
page: 55
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DISCUSSION
Torque teno viruses have several characteristics
(e.g., genomic heterogeneity, great number of
isolates, high prevalence) that play an important
role in the way their analysis must be approached. A
wide range of TTV prevalence has been described
worldwide. Depending on the identification method
used, TTV prevalence may vary from 46.0-62.0% in
Brazil [21,22] to 94.0% in Russia [23]. For TTMV,
prevalence ranges between 48.0% in Norway [24]
and 67.0-72.0% in Brazil [21,22], and TTMDV occurs
in at least 40.0% of the general population [25].
Our results are in accordance with prevalence values
reported for TTVs in other populations, except for
TTMDV, which has a lower prevalence in subjects
selected for this study (31.0%).
Analyzing such heterogeneous genomes represents
a challenge. The high sequence variation of
the genomes, along with high percentage of cytosine
and guanine, make primer design difficult. The
heminested PCR assay designed by Ninomiya et al.
[4] amplifies at least 49 TTV isolates, 20 TTMDV
isolates, and 13 TTMV isolates.
The 10 melting curve patterns on the HRMA suggest
that different viral isolates may be present in our
cohort, or individuals may be coinfected with several
isolates. These may explain the shift of the melting
curve for TTMDV and TTMV samples marked in
Figure 1 and Figure 3.
The melting curve aspect is influenced by the
length, percentage of cytosine and guanine (%GC)
and melting point (Tm) of the amplification products.
The %GC and Tm were calculated in silico for amplicons
produced by five isolates of each virus (Table 2).
The differences between TTV and TTMDV amplicons
are minimal, and an accurate discrimination
is difficult. Moreover, we have run a folding simulation
for each of the amplicons using Quickfold [26] at
65°C (the temperature in the beginning of HRMA) and
observed that each amplicon forms slightly different
secondary structures when denatured. This influences
the melting curve pattern. Subsequent to HRMA, the
resulting amplicons were verified by gel electrophoresis
and no differences were observed (Figure 4).
Another component of the HRMA that influences
the curve patterns is the fluorescent dye. There
are various types of double-stranded DNA (dsDNA)
intercalating dyes with different properties. For
HRMA, the dye must provide detailed information on
the melting behavior of an amplified target. Ideally,
the dye should not bind preferentially to pyrimidines
or purines, change the Tm of the amplicon, or inhibit
DNA amplification.
There are two main types of dyes: saturating
and non saturating dyes. SYBR® Green I is a non
saturating dsDNA intercalating dye and is not usually
recommended for high-resolution melt applications
because at high concentrations, SYBR® Green
I inhibits the DNA polymerase. At low concentrations
SYBR® Green I is able to redistribute from the
melted regions back to the regions of dsDNA, which
results in poor base-difference discrimination [27].
EvaGreen® is a special kind of saturating dye,
so - called “release-on-demand.” For this dye, the fluorescence
is quenched when unbound to DNA, this allows
the use of non saturating dye concentrations, thus
ensuring no PCR inhibition. EvaGreen® improves
the resolution and accuracy of HRMA by increased
fluorescence and lack of redistribution during melting
[28]. Farrar et al. [29] showed that EvaGreen® is more
suitable than SYBR® Green I for HRMA.
Our results show that the best discrimination is
obtained using EvaGreen® (Figure 1 vs. Figures 2
and 3). The differences between the curve patterns
obtained with SYBR® Green I may be due to the
DNA-polymerase activity [SYBR Green Master Mix
(Figure 2) vs. Maxima® SYBR Green qPCR (Figure
3)] and/or dye concentration in the master mixes used
for this study.
As with any technique, HRMA analysis has its
limitations. Fluorescent dyes used in HRMA lack
sequence specificity and can bind to any dsDNA,
including non targets such as primer dimmers and
non specific products, which will bias the results of
melting analysis [28]. In addition, all the PCR components are present at the time of melting analysis
and may have a great impact on the melting curve
shape and position [30].
The amplicons corresponding to TTV, TTMDV,
and TTMV obtained in the assay we used have different
lengths (Table 3). The %GC and also GC distribution
in relation to the ends/center of amplicons
differ due to genomic heterogeneity. These aspects
influence the melting curves’ aspect as well.
Despite these limitations, HRMA is a sensitive
method and provides more information on the amplification
products, such as sequence-dependent shape of
the melting curve and Tm, enabling discrimination of
products with same length but different sequence [31].
Our results showed that HRMA is a rapid method
of detecting human TTVs (HRMA takes approximately
20 min. after second round amplicons are obtained)
compared to the classical PCR-electrophoresis
method, which is more time-consuming (gel preparation,
running and staining). High-resolution melting
analysis provides additional information regarding
amplification products (Tm, melting curve shape)
compared to classic PCR methods followed by gel
electrophoresis, which indicate only the presence or
absence of the target sequence.
In conclusion, due to the advantages of this technique,
HRMA is a rapid and accurate method for
detecting TTVs. Developing new and more sensitive
HRMA assays may lead to easy and accurate detection
of TTV isolates.
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