scholarly journals Identification of Encephalomyocarditis Virus in Clinical Samples by Reverse Transcription-PCR Followed by Genetic Typing Using Sequence Analysis

1998 ◽  
Vol 36 (12) ◽  
pp. 3463-3467 ◽  
Author(s):  
H. Vanderhallen ◽  
F. Koenen
Keyword(s):  
Sequence Analysis ◽  
Reverse Transcription ◽  
Molecular Techniques ◽  
Encephalomyocarditis Virus ◽  
Clinical Samples ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Field Samples ◽  
Pcr Targeting

The objective of the present study was to gain a better understanding of the epidemiology of encephalomyocarditis virus (EMCV) infections in pigs by applying molecular techniques. The diagnostic potential of a reverse transcription-PCR (RT-PCR) targeting 286 nucleotides at the 3′ end of the gene which encodes the viral polymerase was assessed with experimental and field samples. In addition, the use of the amplified sequences for an epidemiological study was evaluated. The heart was clearly shown to be the most suitable organ. The detection limit was determined to be 1 viral particle in 100 mg of heart tissue. The sensitivity and specificity of the assay on the basis of the results obtained in this study were 94 and 100%, respectively. Phylogenetic analysis of the amplified sequences classified EMCVs in two distinct lineages. Group A consists of the reference strain ATCC 129B, all isolates collected between 1991 and 1994 in Belgium in association with reproductive failure, and all Greek isolates. All Belgian isolates collected since the first isolation of EMCV in relation to myocardial failure in fatteners in Belgium group together with the isolates from Cyprus (1996 and 1997), Italy (1986 to 1996), and France (1995) in group B irrespective of their pathogenicity. The analyzed part of the 3D gene differed by 13.0% between Groups A and B. In contrast to the sequence homogeneity of the Belgian isolates collected between 1991 and 1994, molecular diversity, which ranged between 0 and 2%, was observed among the Belgian isolates collected in 1995 and 1996. Among all Greek isolates the diversity ranged between 1 and 8%. However, this diversity does not seem to reflect geographical links between the outbreaks. A RT-PCR for the rapid and specific diagnosis of EMCV in a variety of clinical samples followed by nucleotide sequence analysis proved to be valuable for molecular epidemiological studies.

scholarly journals Verification and diagnostic evaluation of the RealStar® Middle East respiratory syndrome coronavirus (N gene) reverse transcription-PCR kit 1.0

2019 ◽  
Vol 14 (11) ◽  
pp. 941-948 ◽  
Author(s):  
Leonie-Sophie Hecht ◽  
Angeles Jurado-Jimenez ◽  
Markus Hess ◽  
Hussein El Halas ◽  
Gregor Bochenek ◽  
...  
Keyword(s):  
Middle East ◽  
Reverse Transcription ◽  
Diagnostic Procedure ◽  
Middle East Respiratory Syndrome ◽  
Diagnostic Evaluation ◽  
N Gene ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Confirmatory Assay ◽  
Pcr Targeting

Aim: We report the diagnostic evaluation of a confirmatory reverse transcription-PCR (RT-PCR) kit targeting the Middle East respiratory syndrome coronavirus (MERS-CoV) N gene. Material & methods: 33 patient samples from two collections sites in Riyadh, Saudi Arabia, which were pre-characterized via real-time RT-PCR targeting MERS-CoV orf1a and upE, and were tested using the MERS-CoV N gene, as a confirmatory assay. This diagnostic procedure follows a two-step diagnostics scheme, recommended by the WHO. Results: 18/33 samples tested positive, 11/33 tested negative for MERS-CoV RNA and 2/33 showed uncertain results. Conclusion: The results suggest, that the RealStar® MERS-CoV (N gene) RT-PCR kit 1.0 can be considered a suitable and reliable confirmatory assay in combination with the RealStar MERS-CoV RT-PCR kit 1.0 according to the diagnostic scheme recommended by WHO.

scholarly journals Improved Detection of Rhinoviruses in Clinical Samples by Using a Newly Developed Nested Reverse Transcription-PCR Assay

1999 ◽  
Vol 37 (3) ◽  
pp. 524-530 ◽  
Author(s):  
Arno C. Andeweg ◽  
Theo M. Bestebroer ◽  
Martijn Huybreghs ◽  
Tjeerd G. Kimman ◽  
Jan C. de Jong
Keyword(s):  
Reverse Transcription ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Noncoding Regions ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Virus Culture ◽  
Culture Positive

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

scholarly journals Evaluation of a Nested Reverse Transcription-PCR Assay Based on the Nucleoprotein Gene for Diagnosis of Spontaneous and Experimental Bovine Respiratory Syncytial Virus Infections

1999 ◽  
Vol 37 (6) ◽  
pp. 1858-1862 ◽  
Author(s):  
Jean-François Valarcher ◽  
Hervé Bourhy ◽  
Jacqueline Gelfi ◽  
François Schelcher
Keyword(s):  
Respiratory Syncytial Virus ◽  
Reverse Transcription ◽  
Lavage Fluid ◽  
Bovine Respiratory Syncytial Virus ◽  
Virus Infections ◽  
Rt Pcr ◽  
Nucleoprotein Gene ◽  
Reverse Transcription Pcr ◽  
Field Samples ◽  
Syncytial Virus

The first nested reverse transcription (RT)-PCR based on the nucleoprotein gene (n RT-PCR-N) of the bovine respiratory syncytial virus (BRSV) has been developed and optimized for the detection of BRSV in bronchoalveolar lavage fluid cells of calves. This test is characterized by a low threshold of detection (0.17 PFU/ml), which is 506 times lower than that obtained by an enzyme immunosorbent assay (EIA) test (RSV TESTPACK ABBOTT). During an experimental infection of 17 immunocompetent calves less than 3 months old, BRSV RNA could be detected up to 13 days after the onset of symptoms whereas isolation in cell culture was possible only up to 5 days. Compiling results obtained by conventional techniques (serology, antigen detection, and culture isolation) for 132 field samples collected from calves with acute respiratory signs revealed that n RT-PCR-N showed the highest diagnostic sensitivity and very good specificity. This n RT-PCR-N with its long period of detection during BRSV infection thus provides a valuable tool for diagnostic and epidemiological purposes.

scholarly journals Rapid Identification of Nine Microorganisms Causing Acute Respiratory Tract Infections by Single-Tube Multiplex Reverse Transcription-PCR: Feasibility Study

1999 ◽  
Vol 37 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Britta Gröndahl ◽  
Wolfram Puppe ◽  
Andrea Hoppe ◽  
Inka Kühne ◽  
Josef A. I. Weigl ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Reverse Transcription ◽  
Influenza A ◽  
Respiratory Tract Infections ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Tract Infections ◽  
Type 3

Acute respiratory tract infections (ARIs) are leading causes of morbidity and, in developing countries, mortality in children. A multiplex reverse transcription-PCR (RT-PCR) assay was developed to allow in one test the detection of nine different microorganisms (enterovirus, influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenzaviruses type 1 and type 3, adenovirus,Mycoplasma pneumoniae, and Chlamydia pneumoniae) that do not usually colonize the respiratory tracts of humans but, if present, must be assumed to be the cause of respiratory disease. Clinical samples from 1,118 children admitted to the Department of Pediatrics because of an ARI between November 1995 and April 1998 were used for a first clinical evaluation. Detection of one of the microorganisms included in the assay was achieved for 395 of 1,118 (35%) clinical samples, of which 37.5% were RSV, 20% were influenza A virus, 12.9% were adenovirus, 10.6% were enterovirus, 8.1% were M. pneumoniae, 4.3% were parainfluenzavirus type 3, 3.5% were parainfluenzavirus type 1, 2.8% were influenza B virus, and 0.2% were C. pneumoniae. Seasonal variations in the rates of detection of the different organisms were observed, as was expected from the literature. The levels of concordance with the data obtained by commercially available enzyme immunoassays were 95% for RSV and 98% for influenza A. The results show that the multiplex RT-PCR–enzyme-linked immunosorbent assay is a useful and rapid diagnostic tool for the management of children with ARI. Studies of the overall benefit of this method with regard to the use of antibiotics, the use of diagnostic procedures including additional microbiological tests, and hospitalization rate and duration are warranted.

scholarly journals Comparison of a New Gold Immunochromatographic Assay for the Rapid Diagnosis of the Novel Influenza A (H7N9) Virus with Cell Culture and a Real-Time Reverse-Transcription PCR Assay

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Changzhong Jin ◽  
Nanping Wu ◽  
Xiaorong Peng ◽  
Hangping Yao ◽  
Xiangyun Lu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Influenza A ◽  
High Specificity ◽  
High Viral Load ◽  
Immunochromatographic Assay ◽  
Clinical Samples ◽  
H7n9 Virus ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Viral Culture

We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings.

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 Reverse transcription-PCR using a Primer Set Targeting the 3D Region Detects Foot-and-Mouth Disease Virus with High Sensitivity

2018 ◽  
Author(s):  
Tatsuya Nishi ◽  
Toru Kanno ◽  
Nobuaki Shimada ◽  
Kazuki Morioka ◽  
Makoto Yamakawa ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Pcr Assays

AbstractBecause foot-and-mouth disease (FMD) has the potential to spread extensively, methods used for its diagnosis must be rapid and accurate. Therefore, reverse transcription-PCR (RT-PCR) plays an important diagnostic role. Here we designed the primer set FM8/9 to amplify 644 bases of the conserved 3D region of all seven serotypes of FMD virus (FMDV). We compared the performance of RT-PCR assays using FM8/9 with that using the primer set 1F/R targeting the 5’-UTR described in the manual of the World Organization for Animal Health. The detection limits of the RT-PCR assays were determined for 14 strains representing all serotypes. Compared with the sensitivities of the RT-PCR assay using 1F/R, those using FM8/9 were 101-to 104-fold higher for eight strains. To assess the validity of the methods for analyzing clinical samples, sera and saliva samples from pigs and cows infected with FMDV were collected daily and analyzed using the two PCR assays. The FM8/9 assay detected FMDV from all infected pigs and cows for longer times compared with the 1F/R assay, therefore revealing higher sensitivity for the clinical samples. Our results suggest that the FM8/9 RT-PCR assay is highly sensitive and is therefore suitable for the diagnosis of FMD.

scholarly journals Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR

2001 ◽  
Vol 67 (12) ◽  
pp. 5593-5600 ◽  
Author(s):  
Julie Jean ◽  
Burton Blais ◽  
André Darveau ◽  
Ismaı̈l Fliss
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Nucleic Acid Sequence ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Dot Blot ◽  
Reverse Transcription Pcr ◽  
Dot Blot Assay

ABSTRACT A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 102 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 104PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

scholarly journals Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR

2016 ◽  
Vol 55 (3) ◽  
pp. 759-767 ◽  
Author(s):  
Japhette Esther Kembou Tsofack ◽  
Rachel Zamostiano ◽  
Salsabeel Watted ◽  
Asaf Berkowitz ◽  
Ezra Rosenbluth ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Clinical Samples ◽  
Fish Cell ◽  
Rt Pcr ◽  
Fish Cell Lines ◽  
Pcr Assay ◽  
Farmed Fish ◽  
Reverse Transcription Pcr ◽  
Aquaculture Industry ◽  
First Time

ABSTRACT Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.

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.

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