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.
Highlights
Real-time PCR and reverse transcription (RT) PCR techniques are rapid and versatile diagnostic procedures broadly used in clinical virology where there are mostly considered as diagnostic ‘‘gold standards’’ [1]
Monitoring rt-PCR and rt-RTPCR assays and validation of the results rely on the use of relevant external or internal controls (ECs or Internal controls (ICs)) [1,2] and commercial kits including such control systems are being increasingly improved for the molecular diagnosis of a number of pathogens such as HIV, hepatitis viruses, influenza viruses etc
We present results obtained by using T4 and MS2 bacteriophages as ICs in a routine-based evaluation including 8,950 clinical specimens, representing 36 types of samples, submitted for PCR detection of selected viruses including DNA viruses (Herpesviridae, JC and BK viruses, parvovirus B19, adenoviruses) and RNA viruses
Summary
Real-time (rt) PCR and reverse transcription (RT) PCR techniques are rapid and versatile diagnostic procedures broadly used in clinical virology where there are mostly considered as diagnostic ‘‘gold standards’’ [1]. Monitoring rt-PCR and rt-RTPCR assays and validation of the results rely on the use of relevant external or internal controls (ECs or ICs) [1,2] and commercial kits including such control systems are being increasingly improved for the molecular diagnosis of a number of pathogens such as HIV, hepatitis viruses, influenza viruses etc. One of the main strengths of rt-PCR is versatility, which provides the opportunity to set-up ‘‘in-house’’ protocols for specific pathogens. Whilst most commercial kits include both ICs and ECs allowing accurate validation of the results [3], ‘‘home made tests’’ are frequently performed in the absence of ICs and without any possible individual monitoring of each diagnostic reaction. The detection of technical errors or PCR amplification inhibitors is intrinsically impossible if only ECs are used. ECs are usually undistinguishable from the native genome
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