New PCR Test That Recognizes All Human Prototypes of Enterovirus: Application for Clinical Diagnosis

Abstract

We describe a new PCR test (Penter RT-PCR) that recognizes all 64 prototypes of enterovirus. Sixty clinical samples were analyzed in parallel with this Penter RT-PCR and previously described PCR tests: 34 and 32 samples tested positive, respectively. This assay is suitable for use in clinical diagnosis, and its ability to amplify all known serotypes makes it more useful than other consensus PCR tests.Enteroviruses (EVs) are small single-stranded RNA viruses that comprise 64 serotypes recently redistributed into five species (22). They are responsible for a wide variety of clinical manifestations (eruptions and respiratory, ocular, cardiac, and neurological symptoms [8,17]). The PCR test first described almost 10 years ago (3, 9, 18) has become the technique of choice for the diagnosis of these infections, particularly for cerebrospinal fluids (7). The rapidity of diagnosis by PCR has proved to be the determining factor in the management of patients, reducing the cost (by preventing unnecessary use of antibiotics) and duration of hospitalization (15,16). None of the PCR techniques proposed to date, using primers located in the 5Ј noncoding region, recognizes all 64 prototypes of EVs. The primers described by Zoll et al. do not amplify coxsackieviruses A11, A17, A24 or echovirus 16 (23). Furthermore, it is known that EVs exhibit a high degree of intratypic sequence heterogeneity (13). We describe a new PCR method (Penter RT-PCR) that detects all serotypes of EVs.The Penter RT-PCR primers (Penter-1 and 2) were selected from within the 5Ј noncoding region of the EV genome and are 85% identical to known enteroviral RNA sequences. A stair primer PCR system (5, 12) was used. A 420-bp fragment was amplified. This new reverse transcription (RT)-PCR test was performed with all prototypes of EV (obtained from tissue culture or suckling mouse brain), with serotyped field strains, and with frozen clinical samples. These samples were also analyzed by PCR (a technique hereafter referred to as inhouse reference RT-PCR) with primers previously described by Zoll et al. (23) or by Rotbart et al. (19) and by means of the protocols described below.RNA was extracted from prototypes of EV with guanidinium thiocyanate (4, 11). For specimens from patients, RNA was isolated from 140 l of the sample, by using a viral RNA minikit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. RNA from a single extraction was used for both PCR tests, and an aliquot (10 l) of RNA extract was used as a template for RT. The reaction was performed in a final volume of 20 l, and we added 10 l of cDNA to 40 l of reaction mixture for all PCR tests. RNA extraction and RT, cDNA amplification, and amplicon detection were performed in three separate, nonadjacent rooms.For the Penter RT-PCR test, the protocol was as follows: RT was performed with 0.5 M Penter-2, 250 M concentrations (each) of the four deoxynucleoside triphosphates (dNTPs), 50 U of Moloney murine leukemia virus reverse transcriptase (Stratagene, Ozyme, Montigny-le-Bretonne...

New Reverse Transcription-PCR Assay for Rapid and Sensitive Detection of Enterovirus Genomes in Cerebrospinal Fluid Specimens of Patients with Aseptic Meningitis

“…The specificity of the Penter RT-PCR system used for the detection of EV genomes in CSF was demonstrated in a previously published study (2). The sensitivity of the new one-step Penter RT-PCR test was estimated to correspond to 0.05 50% tissue culture infectious doses of poliovirus strain Sabin 2 per 140 ml, according to the protocol previously described by Andréoletti et al Of the 54 CSF specimens selected, 28 (52%) were positive for EV isolation from cell culture whereas 41 (76%) were positive by the in-house RT-PCR assay and 43 (80%) were positive by the new Penter RT-PCR test (52 versus 76 versus 80%; P ϭ 0.003).…”

“…In the present study, we evaluated a new commercially available RT-PCR assay which utilizes the principle of stair primers located in the 5Ј uncoding region to amplify EV genomes (5), detects the EV amplicons in a microwell colorimetric assay within 10 h, and demonstrates the absence of PCR inhibitors by using a positive control tested with a duplicate sample after RNA extraction. This new Penter RT-PCR method has been evaluated in a recent study that demonstrated its ability to recognize all 64 prototypes of human EV strains but not parechovirus 1 and 2 (2). In this study, we used a modified version of this EV amplification method that allows a one-tube RT-PCR amplification of EV genomes.…”

“…An internal control provided by the manufacturer was tested with a duplicate sample after RNA extraction to check the absence of RT-PCR inhibitors in each specimen tested. After chemical denaturation, the single-stranded PCR products obtained from samples and internal controls were separately hybridized by two different biotinylated probes and detected with a streptavidin-peroxidase system on a microtiter plate (2). Results were scored as positive when the optical density (OD) value at 450 nm was above a cutoff value calculated for each serial assay by following the recommendations of the manufacturer (Argène Biosoft) (2).…”

Enterovirus infections following T-cell depleted allogeneic transplants in adults

“…1 Thus, the incidence of enterovirus infections might have been higher if surveillance samples were subjected to more sensitive assays such as RT-PCR, 12,25 but the fact remains that enteroviruses in this study were rarely associated in life-threatening complications. There are a number of possible explanations for these findings.…”

Enterovirus RNA shedding in the genital tract of childbearing-aged women living in Central Africa

“…The commercially available Enterovirus Penter RT-PCR test (''Enterovirus consensus kit new generation'', Argène Biosoft, Varhiles, France) utilizing several pairs of stair primers selected for an annealing in the highly conserved 5 0 untranslated region [Bourlet et al, 2003], was used according to the manufacturer's instructions (Argène Biosoft). This assay has been shown to be able to detect all the 65 human EVs [Bourlet et al, 2003].…”