RNA and DNA Bacteriophages as Molecular Diagnosis Controls in Clinical Virology: A Comprehensive Study of More than 45,000 Routine PCR Tests

Abstract

Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.

Respiratory and gastrointestinal infections at the 2017 Grand Magal de Touba, Senegal: A prospective cohort survey

“…The Multiplex RNA Virus Master Kit (Roche Diagnostics, France) was used to detect influenza A, influenza B, human rhinovirus, human enterovirus, metapneumovirus, respiratory syncytial virus, adenovirus and internal controls MS2 phage by one-step simplex real-time quantitative RT-PCR amplifications [3]. The Multiplex RNA Virus Master Kit (Roche Diagnostics, France) was used to detect influenza A, influenza B, human rhinovirus, human enterovirus, metapneumovirus, respiratory syncytial virus, adenovirus and internal controls MS2 phage by one-step simplex real-time quantitative RT-PCR amplifications [3].…”

Drastic decline of hepatitis E virus detection in domestic pigs after the age of 6 months, Corsica, France

“…Viral RNA was extracted from 200 µl of supernatant using QIAamp Cador Pathogen Mini kit on a QIAcube HT (Qiagen) according to the manufacturer's instructions. An internal control (T4 and MS2 phages) was spiked before extraction, to monitor the extraction and subsequent steps, as described previously (Ninove et al, ). Nucleic acids were eluted in 100µl of RE buffer and stored at −80°C.…”

Absence of Coronaviruses, Paramyxoviruses, and Influenza A Viruses in Seabirds in the Southwestern Indian Ocean

“…The cDNA was diluted 1:2 and stored at 220 C until tested. Before RNA extraction, 10 ml of RNA of the MS2 phage was added to each cloacal sample; after the reverse-transcription step, all samples were tested for cDNA of the MS2 phage (Ninove et al, 2011).…”