Strategic approach to produce low-cost, efficient, and stable competitive internal controls for detection of RNA viruses by use of reverse transcription-PCR

Abstract

Molecular diagnostics based on reverse transcription (RT)-PCR are routinely complicated by the lack of stable internal controls, leading to falsely negative results. We describe a strategy to produce a stable competitive internal control (CIC) based on a Qbeta phage derivative (recombinant Qbeta [rQbeta]) bearing primers KY78 and KY80, which are widely used in the detection of hepatitis C virus (HCV). rQbeta was RNase resistant and stable at 4 degrees C for 452 days in SM medium (0.1 M NaCl, 8 mM MgSO(4).7H(2)O, 50 mM Tris HCl [pH 7.5], 2% gelatin) and for 125 days after lyophilization and reconstitution. rQbeta performance as a CIC was evaluated. rQbeta was added to HCV-positive samples, followed by RNA extraction and a CIC-HCV RT-PCR assay. This method combines RT-PCR, liquid hybridization with nonradioactive probes, and enzyme immunoanalysis. No influence of the CIC on qualitative HCV detection was observed independently of viral load, and results had high concordance with those of commercial kits. In conclusion, we describe a versatile, low-cost alternative strategy to armored RNA technology that can be adapted for detection or real-time applications of any RNA target. Moreover, the CIC reported here is an essential reagent for HCV screening in blood banks in resource-limited settings.

FIG. 1.

Strategy of the rQβ construction as a CIC for an HCV RT-PCR detection assay. (A) QβBst98I and QβNsiI primer sequences. (B) Strategy for KY78 and KY80 insertion into the wild-type Qβ genome. The selected region for the insertion of HCV primer binding sequences lies in the gene coding for the A1 capsid protein of Qβ (GenBank accession number M99039), where Bst98I and NsiI restriction sites are present. Primers QβBst98I and QβNsiI are indicated by arrows. According to our strategy, Primers QβBst98I and QβNsiI would hybridize pBRT7Qβ in the selected region under low-stringency PCR conditions (annealing temperature of 50°C), leading to the generation of a 200-bp chimera fragment containing KY78- and KY80-flanking Qβ sequences. (C) Construction of pBRT7rQβ. Amplicons containing heterologous sequences of KY78 and KY80 were purified and subcloned into the pGEM-T-Easy cloning vector, creating plasmid pQβp′. Each plasmid, pQβp′ and pBRT7Qβ, was digested with Bst98I and NsiI restriction enzymes. The 200-bp chimera fragment from pQβp′ was purified from the vector and ligated into pBRT7Qβ derived from the 200-bp wild-type fragment, creating pBRT7rQβ. (D) A1 region sequence of the pBRT7rQβ derivative. The correct construction of pBRT7rQβ was confirmed by sequencing of the A1 region, where heterologous primers were introduced. Note that no nucleotides were added or deleted with respect wild-type Qβ to avoid alterations in phage RNA folding and assembly. The Qβ sequence where the VQβ02 probe hybridizes is shown in italics. Wild-type Qβ sequences are shown in capital letters, and heterologous sequences (KY78 and KY80) are shown in lowercase letters. Bst98I and NsiI restriction sites are underlined.

FIG. 2.

Steps of the CIC-HCV RT-PCR assay. (A) RNA extraction. A fixed amount of the CIC (rQβ) was added to the plasma sample, followed by RNA extraction. (B) RT-PCR. The CIC (rQβ) and HCV were first retrotranscribed using the antisense HCV-specific primer biotinylated in the 5′ end (B-KY78). After the RT reaction, biotinylated cDNAs were generated from both targets. Sense HCV primer (KY80) was added, and PCR amplification was performed, leading to the generation of biotinylated amplicons of 244 bp (HCV) and 200 bp (rQβ). (C) Liquid hybridization. Biotinylated amplicons were denatured and then hybridized to specific HCV (F-KY88) or rQβ (F-VQβ02) probes at the corresponding hybridization temperatures (T_H). Since both probes contain fluorescein in their 5′ ends (F−), biotinylated-fluoresceinated hybrids were obtained after this procedure. (D) EIA. Biotinylated-fluoresceinated hybrids were captured in streptavidin-coated microplate wells. Colorimetric detection was performed with a monospecific anti-fluorescein antibody conjugated with horseradish peroxidase (aFP), TMB substrate, and H₂O₂. Reactive samples turn from colorless (C) to blue (B) by the oxidation of TMB substrate. The reaction was stopped by the addition of H₂SO₄, and a color change from blue to yellow occurred if the sample gave a positive result. The end-point OD was read at 450 nm in a microplate reader.