Improved diagnosis on a daily basis of enterovirus meningitis using a one-step real-time RT-PCR assay

被引:20
作者
Archimbaud, C [1 ]
Mirand, A [1 ]
Chambon, M [1 ]
Regagnon, C [1 ]
Bailly, JL [1 ]
Peigue-Lafeuille, N [1 ]
Henquell, C [1 ]
机构
[1] Ctr Hosp Univ, Fac Med, Lab Virol Med, F-63000 Clermont Ferrand, France
关键词
cerebrospinal fluid specimens; LightCycler; SYBR Green; conventional in-house RT-PCR; inter- and intra-assay;
D O I
10.1002/jmv.20217
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The detection of the enterovirus genome in cerebrospinal fluid (CSF) by PCR techniques has proved to be more sensitive than traditional cell culture for the diagnosis of enterovirus meningitis. However, PCR assays are time consuming and labor intensive, particularly if separate hybridization steps are used to confirm the specificity of positive findings. The aim of this study was to develop a one-step real-time RT-PCR assay with LightCycler (LC) technology that was sensitive, rapid, and easy to perform in routine practice. The enterovirus detection limit was determined by testing 10-fold limiting dilution series of cell culture stocks with the echovirus 25 (E-25) prototype strain and with the third European Union Quality Control Concerted Action (EU-QCCA) enterovirus proficiency panel. A total of 100 CSF specimens were investigated in a comparative study. With the E-25 strain, the detection limit of the real-time assay was 286 TCID50/ml (50% tissue culture infective dose). When samples of the EUQCCA panel were tested, our assay gave identical results (detection limit down to 3.6 TCID50/ml) to those of the reference laboratory, which used one-step RT-PCR assay. When CSF specimens were tested, there was a correlation between the real-time assay and the conventional in-house assay in 96 of 100 CSFs tested. This one-step real-time assay allows rapid enterovirus detection in CSF since results are obtained in 3 hr as against 36 hr with the "in-house" RT-PCR assay. This new assay is now being used in routine practice, and allows diagnosis on a daily basis.
引用
收藏
页码:604 / 611
页数:8
相关论文
共 26 条
[1]  
Andréoletti L, 1998, J CLIN MICROBIOL, V36, P589
[2]   Molecular evidence of persistent echovirus 13 meningoencephalitis in a patient with relapsed lymphoma after an outbreak of meningitis in 2000 [J].
Archimbaud, C ;
Bailly, JL ;
Chambon, M ;
Tournilhac, O ;
Travade, P ;
Peigue-Lafeuille, H .
JOURNAL OF CLINICAL MICROBIOLOGY, 2003, 41 (10) :4605-4610
[3]   NEW METHOD FOR THE EXTRACTION OF VIRAL-RNA AND DNA FROM CEREBROSPINAL-FLUID FOR USE IN THE POLYMERASE CHAIN-REACTION ASSAY [J].
CASAS, I ;
POWELL, L ;
KLAPPER, PE ;
CLEATOR, GM .
JOURNAL OF VIROLOGICAL METHODS, 1995, 53 (01) :25-36
[4]   Circulation of enteroviruses and persistence of meningitis cases in the winter of 1999-2000 [J].
Chambon, M ;
Archimbaud, C ;
Bailly, JL ;
Henquell, C ;
Regagnon, C ;
Charbonné, F ;
Peigue-Lafeuille, H .
JOURNAL OF MEDICAL VIROLOGY, 2001, 65 (02) :340-347
[5]  
Chandler DP, 1998, APPL ENVIRON MICROB, V64, P669
[6]   Development and evaluation of a 'real-time' RT-PCR for the detection of enterovirus and parechovirus RNA in CSF and throat swab samples [J].
Corless, CE ;
Guiver, M ;
Borrow, R ;
Edwards-Jones, V ;
Fox, AJ ;
Kaczmarski, EB ;
Mutton, KJ .
JOURNAL OF MEDICAL VIROLOGY, 2002, 67 (04) :555-562
[7]   Prospective analysis of 61 cases of enteroviral meningitis:: interest of systematic genome detection in cerebrospinal fluid irrespective of cytologic examination results [J].
Henquell, C ;
Chambon, M ;
Bailly, JL ;
Alcaraz, S ;
De Champs, C ;
Archimbaud, C ;
Labbé, A ;
Charbonné, F ;
Peigue-Lafeuille, H .
JOURNAL OF CLINICAL VIROLOGY, 2001, 21 (01) :29-35
[8]   Quantitation of HCV RNA using real-time PCR and fluorimetry [J].
Komurian-Pradel, F ;
Paranhos-Baccalà, G ;
Sodoyer, M ;
Chevallier, P ;
Mandrand, B ;
Lotteau, V ;
André, P .
JOURNAL OF VIROLOGICAL METHODS, 2001, 95 (1-2) :111-119
[9]  
LIM KA, 1960, J IMMUNOL, V84, P309
[10]  
MODLIN JF, 1997, ADV PEDIAT INFECT DI, V12, P155