Dual-Emission Fluorescence Resonance Energy Transfer (FRET) Real-Time PCR Differentiates Feline Immunodeficiency Virus Subtypes and Discriminates Infected from Vaccinated Cats

Abstract

Feline immunodeficiency virus (FIV) is among the most common infectious agents of cats. Five well-characterized

Identification of feline immunodeficiency virus subtype-B on St. Kitts, West Indies by quantitative PCR

“…Based on the PCR amplification efficiencies and the Tm conditions as demonstrated during PCR amplification and melting curve analysis of synthesized amplicons of FIV subtypes A, B, C, D and E [12], all four FIV isolates from St. Kitts were recognized as FIV subtype-B. The amplification curve for FIV subtype-B was less vigorous than for subtypes A and C in the fluorescence F4/F1 channel [12], and no peak appeared in the fluorescence channel F6/F1.…”

“…The design of the primers and probes, real-time PCR platform, and high-resolution melting curve analysis were performed as described [12][13][14]. The FIV copy number was determined by FRET-PCR performed in a six-channel LightCycler 2.0 Instrument with Software version 4.1 (Roche Molecular Biochemicals, Indianapolis, IN, USA).…”

“…Since FIV subtype D is substantially different from other subtypes, the probes F1-D (6-FAM-TAC TTC GTT GTC CCG TAC CTA CAG CA-Phosphate) and F6-D (CCA TCT AAA ATT TCC TTC CCC GAA CTT C-CY5.5, λ = 705 nm) were used to detect FIV subtype-D. Probes F1-ABCE (6-FAM-TAC TCT TSS CCC CTA CTC CTA CAG CA-6-FAM) and F4-ABCE (LC Red 640-CAT TAC TAC ATC TYT TWA TGG CCA YTT TCC A-Phosphate, λ = 640 nm) were used to detect subtypes A, B, C, and E [12].…”

“…The plasmid was restricted with HindIII (Promega, Madison, WI, USA) and quantified by PicoGreen DNA fluorescence assay (Molecular Probes, Eugene, OR, USA) for preparation of quantitative standards. The melting curve for the annealing of the PCR product was determined by monitoring the fluorescence from 37°C to 85°C, and the temperature transition rate of 0.2°C per second was applied [12,14]. The fluorescence ratios F4/F1 and F6/F1 were analyzed, and the negative first derivatives of F4/F1 and F6/F1 were evaluated for determination of probe T m.…”

“…Recently, a dual-emission fluorescence resonance energy transfer (FRET) real-time PCR has been described which detects FIV in blood and can differentiate the FIV subtypes [12]. In this report we describe the use of the FRET PCR to confirm the presence of FIV in cats on St Kitts and to establish which FIV subtypes occur.…”