scholarly journals Evaluation of a Commercially Available Reverse Transcription-PCR Assay for Diagnosis of Enteroviral Infection in Archival and Prospectively Collected Cerebrospinal Fluid Specimens

1998 ◽  
Vol 36 (6) ◽  
pp. 1741-1745 ◽  
Author(s):  
Francisco Pozo ◽  
Inmaculada Casas ◽  
Antonio Tenorio ◽  
Gloria Trallero ◽  
Jose M. Echevarria
Keyword(s):  
Cell Culture ◽  
Cerebrospinal Fluid ◽  
Aseptic Meningitis ◽  
Reverse Transcription ◽  
Diagnostic Systems ◽  
Rt Pcr ◽  
Enteroviral Infection ◽  
Reverse Transcription Pcr ◽  
Sporadic Cases ◽  
Pcr Method

A commercially available reverse transcription (RT)-PCR method (AMPLICOR EV; Roche Diagnostic Systems, Inc., Branchburg, N.J.) was evaluated for detection of enteroviruses in cerebrospinal fluid from patients with neurological disease. This assay was compared with virus isolation in cell culture and an in-house RT-PCR method designed with a nonoverlapping region of the enteroviral genome. A panel of 200 cerebrospinal fluid specimens prospectively collected from patients with a wide variety of neurological symptoms, including 50 patients involved in three different outbreaks of acute aseptic meningitis, was assayed. A second panel of 97 archived cerebrospinal fluid specimens, stored for 2 to 5 years, from patients with aseptic meningitis associated with several enterovirus outbreaks was also studied. From the first panel, enteroviruses were detected in 13 of 50 specimens by cell culture (26%), in 43 of 50 specimens by AMPLICOR EV (86%), and in 46 of 50 specimens by the in-house assay (92%) from patients with aseptic meningitis associated with outbreak and 1 of 29, 3 of 29, and 4 of 29 specimens, respectively, from sporadic cases of aseptic meningitis. The remaining 121 cerebrospinal fluid specimens from patients with other neurological syndromes were negative by all tests. From the second panel, enteroviral RNA was detected by the AMPLICOR test (31 of 97 specimens, 32%) and the in-house assay (39 of 97 specimens, 40%). According to our results, patients with aseptic meningitis should be analyzed for enteroviral infection in cerebrospinal fluid by RT-PCR methods, and the AMPLICOR EV test is a suitable tool for performing such studies. Archival cerebrospinal fluid specimens are less suitable for evaluation of the performance of RT-PCR methods designed for enterovirus detection.

scholarly journals Detection by PCR of Enteroviruses in Cerebrospinal Fluid during a Summer Outbreak of Aseptic Meningitis in Switzerland

1998 ◽  
Vol 36 (9) ◽  
pp. 2408-2412 ◽  
Author(s):  
Meri Gorgievski-Hrisoho ◽  
Jean-Daniel Schumacher ◽  
Nevenka Vilimonovic ◽  
Daniel Germann ◽  
Lukas Matter
Keyword(s):  
Cell Culture ◽  
Cerebrospinal Fluid ◽  
Aseptic Meningitis ◽  
Diagnostic Techniques ◽  
Rt Pcr ◽  
Echovirus 30 ◽  
Reverse Transcription Pcr ◽  
Viral Culture ◽  
Test Kit ◽  
Virus Culture

Enteroviruses (EV) are among the most common causes of aseptic meningitis. Standard diagnostic techniques are often too slow and lack sensitivity to be of clinical relevance. EV RNA can be detected within 5 h by a commercially available reverse transcription-PCR (RT-PCR) test kit. Cerebrospinal fluid (CSF) samples from 68 patients presenting with aseptic meningitis during a summer outbreak in Switzerland were examined in parallel with cell culture and commercial RT-PCR. RT-PCR was positive in all 16 CSF specimens positive by cell culture (100%). In addition, 42 of 52 (80%) CSF samples negative by cell culture were PCR positive. In 26 of these 42 (62%) patients, viral culture from other sites (throat swab or stool) was also positive. The CSF virus culture took 3 to 7 days to become positive. Echovirus 30 was the type most often isolated in this outbreak. The sensitivity of CSF RT-PCR based on clinical diagnosis during this aseptic meningitis outbreak in patients with negative bacterial culture results was 85%, i.e., considerably higher than the sensitivity of CSF virus culture (24%). We conclude that this commercial RT-PCR assay allows a positive diagnosis with minimal delay and may thus influence clinical decisions.

scholarly journals Detection and Identification of Mammalian Reoviruses in Surface Water by Combined Cell Culture and Reverse Transcription-PCR

2001 ◽  
Vol 67 (7) ◽  
pp. 3016-3020 ◽  
Author(s):  
Michael L. Spinner ◽  
George D. Di Giovanni
Keyword(s):  
Cell Culture ◽  
Surface Water ◽  
Sequence Analysis ◽  
Reverse Transcription ◽  
Mammalian Species ◽  
Sampling Site ◽  
Water Sources ◽  
Sequence Diversity ◽  
Rt Pcr ◽  
Reverse Transcription Pcr

ABSTRACT Reoviruses are a common class of enteric viruses capable of infecting a broad range of mammalian species, typically with low pathogenicity. Previous studies have shown that reoviruses are common in raw water sources and are often found along with other animal viruses. This suggests that in addition to the commonly monitored enteroviruses, reoviruses might serve as an informative target for monitoring fecal contamination of drinking water sources. Mammalian reoviruses were detected and identified by a combined cell culture–reverse transcription-PCR (RT-PCR) assay with novel primers targeting the L3 gene that encodes the λ3 major core protein. Five of 26 (19.2%) cytopathic effect-positive cell culture lysates inoculated with surface water were positive for reoviruses by RT-PCR. DNA sequence analysis of RT-PCR products revealed significant sequence diversity among isolates, which is consistent with the sequence diversity among previously characterized mammalian reoviruses. Sequence analysis revealed persistence of a reovirus genotype at a single sampling site, while a sample from another site contained two different reovirus genotypes.

scholarly journals Detection of Flavescence Dorée Phytoplasma in Grapevine by Reverse-Transcription PCR

Plant Disease ◽  
2007 ◽  
Vol 91 (11) ◽  
pp. 1496-1501 ◽  
Author(s):  
P. Margaria ◽  
C. Rosa ◽  
C. Marzachì ◽  
M. Turina ◽  
S. Palmano
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Diagnostic Methods ◽  
Kappa Index ◽  
Rt Pcr ◽  
Flavescence Dorée ◽  
Reverse Transcription Pcr ◽  
Novel Method ◽  
Pcr Method ◽  
Polymerase Chain

Flavescence dorée (FD) is the most serious phytoplasma disease of grapevine. This report describes a novel method of detecting FD phytoplasma based on reverse-transcription polymerase chain reaction (RT-PCR) on 16S ribosomal RNA (16SrRNA) which will greatly improve mass screening of infected grapevines. A rapid protocol for extracting sap from whole leaves or midveins and successive one-tube amplification by RT-PCR was applied to grapevine samples with or without symptoms collected from different areas of Piedmont (northwestern Italy). Results were compared with those obtained using one of the current diagnostic methods that utilizes nested PCR on phytoplasma DNA-enriched preparations. A Cohen's kappa index of 0.76 indicated a substantial agreement between the two sets of results. The RT-PCR method has the advantage of being a rapid, reliable, and sensitive assay for large-scale screening of grapevines.

scholarly journals Molecular detection and characterisation of mumps virus in cerebrospinal fluid in a Gauteng laboratory

2016 ◽  
Vol 31 (1) ◽  
pp. 29-31
Author(s):  
Marieke Brauer ◽  
Marianne Wolfaardt ◽  
Lynne M. Webber ◽  
Maureen B. Taylor
Keyword(s):  
Polymerase Chain Reaction ◽  
Phylogenetic Analysis ◽  
Cerebrospinal Fluid ◽  
Aseptic Meningitis ◽  
Reverse Transcription ◽  
Molecular Detection ◽  
Mumps Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

The study aimed to determine the presence of mumps virus (MuV) in cerebrospinal fluid (CSF) specimens and to genetically characterise detected MuV strains. A real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the MuV F gene, and characterisation was performed by sequencing of the SH gene. Mumps virus was detected in 1.2% (3/260) of specimens. Phylogenetic analysis of one MuV strain revealed that it clustered with the Jeryl-Lynn and RIT4385 vaccine strains. As far as the authors could ascertain this is the first study to provide viral proof that these vaccine-like strains may be associated with aseptic meningitis.

scholarly journals Detection and Identification of Human Parainfluenza Viruses 1, 2, 3, and 4 in Clinical Samples of Pediatric Patients by Multiplex Reverse Transcription-PCR

2000 ◽  
Vol 38 (3) ◽  
pp. 1191-1195 ◽  
Author(s):  
Jose C. Aguilar ◽  
María P. Pérez-Breña ◽  
María L. García ◽  
Nieves Cruz ◽  
Dean D. Erdman ◽  
...  
Keyword(s):  
Cell Culture ◽  
Reverse Transcription ◽  
Pediatric Patients ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Parainfluenza Viruses ◽  
Cell Culture Isolation ◽  
Human Parainfluenza Viruses

We describe a multiplex reverse transcription-PCR (m-RT-PCR) assay that is able to detect and differentiate all known human parainfluenza viruses (HPIVs). Serial dilution experiments with reference strains that compared cell culture isolation and m-RT-PCR showed sensitivities ranging from 0.0004 50% tissue culture infective dose (TCID50) for HPIV type 4B (HPIV-4B) to 32 TCID50s for HPIV-3. As few as 10 plasmids containing HPIV PCR products could be detected in all cases. When 201 nasopharyngeal aspirate specimens from pediatric patients hospitalized for lower respiratory illness were tested, m-RT-PCR assay detected 64 HPIVs (24 HPIV-3, 23 HPIV-1, 10 HPIV-4, and 7 HPIV-2), while only 42 of them (21 HPIV-1, 14 HPIV-3, 6 HPIV-2, and 1 HPIV-4 isolates) grew in cell culture. Our m-RT-PCR assay was more sensitive than either cell culture isolation or indirect immunofluorescence with monoclonal antibodies for the detection of HPIV infections. Also, HPIV-4 was more frequently detected than HPIV-2 in this study, suggesting that it may have been underestimated as a lower respiratory tract pathogen because of the insensitivity of cell culture.

Detection of Canine Distemper Virus Nucleoprotein RNA by Reverse Transcription-PCR Using Serum, Whole Blood, and Cerebrospinal Fluid from Dogs with Distemper

1999 ◽  
Vol 37 (11) ◽  
pp. 3634-3643 ◽  
Author(s):  
A. L. Frisk ◽  
M. König ◽  
A. Moritz ◽  
W. Baumgärtner
Keyword(s):  
Cerebrospinal Fluid ◽  
Reverse Transcription ◽  
Whole Blood ◽  
Canine Distemper Virus ◽  
Detection System ◽  
Nucleotide Sequence Analysis ◽  
Restriction Enzyme Digestion ◽  
Canine Distemper ◽  
Rt Pcr ◽  
Reverse Transcription Pcr

Reverse transcription-PCR (RT-PCR) was used to detect canine distemper virus (CDV) nucleoprotein (NP) RNA in serum, whole blood, and cerebrospinal fluid (CSF) samples from 38 dogs with clinically suspected distemper. Results were correlated to clinical findings, anti-CDV neutralizing antibody titers, postmortem findings, and demonstration of CDV NP antigen by immunohistochemistry. The specificity of the RT-PCR was ensured by amplification of RNA from various laboratory CDV strains, restriction enzyme digestion, and Southern blot hybridization. In 29 of 38 dogs, CDV infection was confirmed by postmortem examination and immunohistochemistry. The animals displayed the catarrhal, systemic, and nervous forms of distemper. Seventeen samples (serum, whole blood, or CSF) from dogs with distemper were tested with three sets of primers targeted to different regions of the NP gene of the CDV Onderstepoort strain. Expected amplicons were observed in 82, 53, and 41% of the 17 samples, depending upon the primer pair used. With the most sensitive primer pair (primer pair I), CDV NP RNA was detected in 25 of 29 (86%) serum samples and 14 of 16 (88%) whole blood and CSF samples from dogs with distemper but not in body fluids from immunohistochemically negative dogs. Nucleotide sequence analysis of five RT-PCR amplicons from isolates from the field revealed few silent point mutations. These isolates exhibited greater homology to the Rockborn (97 to 99%) than to the Onderstepoort (95 to 96%) CDV strain. In summary, although the sensitivity of the RT-PCR for detection of CDV is strongly influenced by the location of the selected primers, this nucleic acid detection system represents a highly specific and sensitive method for the antemortem diagnosis of distemper in dogs, regardless of the form of distemper, humoral immune response, and viral antigen distribution.

scholarly journals Evaluation of a Commercial DNA Enzyme Immunoassay for Detection of Enterovirus Reverse Transcription-PCR Products Amplified from Cerebrospinal Fluid Specimens

2000 ◽  
Vol 38 (11) ◽  
pp. 4260-4261 ◽  
Author(s):  
Pampee P. Young ◽  
Richard S. Buller ◽  
Gregory A. Storch
Keyword(s):  
Cerebrospinal Fluid ◽  
Enzyme Immunoassay ◽  
Optical Density ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Clinical Laboratories ◽  
Pcr Products ◽  
Dna Enzyme Immunoassay ◽  
Culture Positive

We evaluated the DiaSorin DNA enzyme immunoassay (DEIA) kit for detection of enteroviral reverse transcription-PCR (RT-PCR) products amplified from cerebrospinal fluid. By use of an optical density of 0.05 as the absorbance cutoff, 35% of 198 specimens were PCR positive, whereas 16% were culture positive. DEIA was rapid and sensitive and can help implement enterovirus RT-PCR in clinical laboratories.

scholarly journals Quantification of Clostridium botulinumToxin Gene Expression by Competitive Reverse Transcription-PCR

2000 ◽  
Vol 66 (4) ◽  
pp. 1423-1428 ◽  
Author(s):  
S. McGrath ◽  
J. S. G. Dooley ◽  
R. W. Haylock
Keyword(s):  
Gene Expression ◽  
Reverse Transcription ◽  
Botulinum Neurotoxin ◽  
Food Products ◽  
Toxin Production ◽  
Mouse Bioassay ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Challenge Testing ◽  
Pcr Method

ABSTRACT Clostridium botulinum produces a characteristic botulinum neurotoxin which can cause an often fatal neuroparalytic condition known as botulism. Although food-borne botulism is rare, critical screening by food companies is necessary to ensure that food products are safe. At present, the food industry assesses the risks of botulinum neurotoxin production by challenge testing to check any new food products and to check the efficacy of new storage regimes. Challenge testing involves artificial introduction of defined strains of microorganisms into food, and microbial growth and possible toxin production are then monitored. Botulinum toxin is normally analyzed by using the mouse bioassay. However, the mouse bioassay is expensive, slow, and politically sensitive because of animal rights issues. In this paper we describe adaptation of a new assay, competitive reverse transcription-PCR (RT-PCR), to monitor botulinum neurotoxin production. This method accurately measures the level of toxin-encoding mRNA inC. botulinum cells. Measurement of mRNA should provide a good indication of gene expression as mRNA is turned over rapidly in bacterial cells. In addition, the method is rapid, specific, and sensitive. The competitive RT-PCR method was developed to examineC. botulinum E VH toxin gene expression and was used to investigate the level of toxin production by C. botulinum E VH when the organism was grown in two different types of broth. The results which we obtained with the competitive RT-PCR method demonstrated that this method is more rapid and more sensitive than the mouse bioassay.

scholarly journals Reverse Transcription-Booster PCR for Detection of Noroviruses in Shellfish

2004 ◽  
Vol 70 (10) ◽  
pp. 6329-6332 ◽  
Author(s):  
Dario De Medici ◽  
Luciana Croci ◽  
Elisabetta Suffredini ◽  
Laura Toti
Keyword(s):  
Reverse Transcription ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Pcr Method ◽  
Higher Sensitivity

ABSTRACT The methods commonly used for norovirus (NV) detection are based on reverse transcription-PCR (RT-PCR) followed by confirmation of the amplified sequence. To increase sensitivity, an RT-booster PCR was developed. The proposed method showed an increase in sensitivity at least 2 log units for all the NV strains tested compared with the standard RT-PCR method. Higher sensitivity was confirmed in tests on experimentally and naturally contaminated shellfish.

Sign in / Sign up

Export Citation Format

Share Document