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

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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