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Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens.
J Clin Virol. 2006 Mar; 35(3):278-84.JC

Abstract

BACKGROUND

Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis.

OBJECTIVE

To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens.

STUDY DESIGN

After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay.

RESULTS

Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID50 for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the "gold standard" were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k=0.80).

CONCLUSIONS

The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management.

Authors+Show Affiliations

Laboratory of Human and Molecular Virology, University Hospital, Avenue G. Clemenceau, 14033 Caen Cedex, France. petitijean-j@chu-caen.frNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Evaluation Study
Journal Article

Language

eng

PubMed ID

16214398

Citation

Petitjean, J, et al. "Development and Evaluation of a Real-time RT-PCR Assay On the LightCycler for the Rapid Detection of Enterovirus in Cerebrospinal Fluid Specimens." Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology, vol. 35, no. 3, 2006, pp. 278-84.
Petitjean J, Vabret A, Dina J, et al. Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens. J Clin Virol. 2006;35(3):278-84.
Petitjean, J., Vabret, A., Dina, J., Gouarin, S., & Freymuth, F. (2006). Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens. Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology, 35(3), 278-84.
Petitjean J, et al. Development and Evaluation of a Real-time RT-PCR Assay On the LightCycler for the Rapid Detection of Enterovirus in Cerebrospinal Fluid Specimens. J Clin Virol. 2006;35(3):278-84. PubMed PMID: 16214398.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens. AU - Petitjean,J, AU - Vabret,A, AU - Dina,J, AU - Gouarin,S, AU - Freymuth,F, Y1 - 2005/10/06/ PY - 2004/05/24/received PY - 2005/02/27/accepted PY - 2005/10/11/pubmed PY - 2006/4/28/medline PY - 2005/10/11/entrez SP - 278 EP - 84 JF - Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology JO - J Clin Virol VL - 35 IS - 3 N2 - BACKGROUND: Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis. OBJECTIVE: To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens. STUDY DESIGN: After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay. RESULTS: Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID50 for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the "gold standard" were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k=0.80). CONCLUSIONS: The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management. SN - 1386-6532 UR - https://www.unboundmedicine.com/medline/citation/16214398/Development_and_evaluation_of_a_real_time_RT_PCR_assay_on_the_LightCycler_for_the_rapid_detection_of_enterovirus_in_cerebrospinal_fluid_specimens_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1386-6532(05)00243-X DB - PRIME DP - Unbound Medicine ER -