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

MATERIALS AND METHODS

Samples and DNA Extraction. A total of 113 healthy subjects from Bucharest, Romania, ranging in age from 19 to29 years, were selected for this study. Blood and saliva samples from 13 subjects were used for protocol optimization, whereas saliva samples (n = 100) were used to estimate the TTVs prevalence. All subjects gave written informed consent to participate in this study according to the Helsinki Declaration of 1975, as revised in 1983, concerning the ethical principles for medical research involving human subjects. DNA was extracted from each blood sample using four different commercial kits (recommended for DNA extraction from eukaryotic cells): two spin-column-based protocols, AxyPrep™ Blood Genomic DNA Miniprep Kit (Axygen, Union City, CA, USA) and Ron’s Blood DNA Mini Kit (Bioron, Ludwigshafen, Germany), and two pro-tocols without spin-column. Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA), Pronto® DNA Extraction Kit (Pronto Diagnostics, Rehovot, Israel). The DNA was extracted according to the manufacturer’s instructions from 250 mL of blood. A one-tube purification method, OTP reagent (Bioron), was used for DNA extrac-tion from saliva. Polymerase Chain Reaction. The detection of the three TTVs was realized using a two-step heminested- PCR protocol as described by Ninomiya et al. [4]. Each DNA sample was PCR amplified using three DNA-polymerases: GoTaq® DNA Polymerase (Promega), Immolase™ DNA Polymerase (Bioline, Luckenwalde, Germany), and Dry PCR Master Mix (blue-stain) (Bioron). For the first step, the amplification reaction was car-ried out in a total volume of 15 mL containing 1 × PCR buffer solution, 1.5 mmol/L MgCl2, 100 mmol/L dNTP mix, 0.5 mmol/L of each primer, 0.6 units of DNA-polymerase and 1.5 mL of genomic DNA extract. The second step consisted of three different reactions for the specific amplification of the viral species. For the second step, 1 mL of amplicon from the first round of PCR was used as a template. The amplification programs and primers can be found in Table 1. The amplicons of the second PCR were resolved by 2.0% (w/v) agarose gel and were electropho-resed at 5 V/cm for 20 min. The gel was visualized under UV light after ethidium bromide staining. High-Resolution Melting Analysis. The samples co-infected with all three TTVs were further analyzed by HRMA using three commercial kits and two different fluorescent dyes: SensiMix® with EvaGreen® (Bioline), Invitek SYBR Green Master Mix (Invitek, Berlin, Germany) and Maxima® SYBR Green qPCR (Fermentas, Glen Burnie, MD, USA). The amplification reaction was carried out in a total volume of 20 mL and used 2 mL of second-round amplicons as template DNA. High-resolution melting analysis was performed in a Rotor-Gene 6000 series instrument (Corbett Research, Sydney, NSW, Australia) using software version 1.7. Melting curves were generated by increasing the tem-perature from 65°C to 90°C ramping by 0.1 degrees per step, with a gap of 2 seconds between steps. For the HRMA normalization regions of 75- 77°C and 88-90°C were applied.



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