Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics|
Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão & Krieger, Marco Aurélio
BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections.
In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human
immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary
to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test.
OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as
IC for the diagnosis of RNA viruses.
METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were
produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC
in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated
HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of
MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection.
FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV
amplification performance, including the extraction, reverse transcription, amplification and detection steps, without
compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this
strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An
attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than
one pathogen, increasing its applicability for diagnosing different RNA viruses.
diagnostics; Real-time PCR; internal control; HCV; RNA viruses