scholarly journals Evaluation of six TaqMan RT-rtPCR kits on two thermocyclers for the reliable detection of rabies virus RNA

2018 ◽  
Vol 31 (1) ◽  
pp. 47-57 ◽  
Author(s):  
Evelyne Picard-Meyer ◽  
Carine Peytavin de Garam ◽  
Jean Luc Schereffer ◽  
Emmanuelle Robardet ◽  
Florence Cliquet
Keyword(s):  
Real Time ◽  
Rabies Virus ◽  
Fluorescent Antibody ◽  
Animal Health ◽  
Antibody Test ◽  
Virus Rna ◽  
Field Samples ◽  
Rapid Amplification ◽  
World Organization ◽  
Positive Results

Rabies is diagnosed postmortem in animals, based on tests prescribed by the World Organization for Animal Health (OIE), such as the fluorescent antibody test, the direct rapid immunohistochemistry test, or pan-lyssavirus PCR assays. Several reverse-transcription real-time PCR (RT-rtPCR) methods have been developed and validated for rapid and accurate detection of lyssaviruses. We evaluated the performance of 6 TaqMan RT-rtPCR kits using different commercial master mixes and 2 real-time thermocyclers. Changing the master mix overall did not influence the TaqMan RT-rtPCR performance, regardless of the thermocycler used. The limits of detection at the 95% confidence level were 18.1–25.8 copies/µL for the Rotor-Gene Q MDx thermocycler and 16.7–21.5 for the Mx3005P thermocycler. Excellent repeatability was demonstrated for rabies virus (RABV) RNA samples of 100, 50, and 25 copies/µL regardless of the thermocycler used. RABV field samples ( n = 35) isolated worldwide gave positive results using the most efficient of the 6 kits tested, with a copy number of 6.03 × 102 to 6.78 × 107 RNA copies per reaction. The TaqMan RT-rtPCR assay provides sensitive and rapid amplification of RABV RNA.

scholarly journals Detection of multiple strains of rabies virus RNA using primers designed to target Mexican vampire bat variants

2005 ◽  
Vol 133 (5) ◽  
pp. 927-934 ◽  
Author(s):  
E. LOZA-RUBIO ◽  
E. ROJAS-ANAYA ◽  
V. M. BANDA-RUÍZ ◽  
S. A. NADIN-DAVIS ◽  
B. CORTEZ-GARCÍA
Keyword(s):  
Rabies Virus ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
N Gene ◽  
Pcr Assay ◽  
Virus Rna ◽  
Geographical Regions ◽  
Polymerase Chain ◽  
Multiple Strains ◽  
Vampire Bat

A reverse transcription–polymerase chain reaction (RT–PCR), that uses primers specifically designed to amplify a portion of the N gene of vampire bat strains of rabies that circulate in Mexico, but also recognizing most of the rabies variants circulating in endemic areas, was established. This standardized PCR assay was able to detect viral RNA in tenfold serial dilutions up to a 107 dilution using stock virus at an original titre of 107·5 LD50. The assay was highly specific for rabies virus. Forty different rabies isolates recovered from different species and geographical regions in the country were diagnosed as positive and negative by the fluorescent antibody test (FAT). These same samples were re-examined by both PCR and the mouse inoculation test (MIT). Compared with MIT the PCR exhibited an epidemiological sensitivity of 86% and a specificity of 91% while its positive predictive value was 96%.

scholarly journals Quantification of Rabies Virus by Real Time PCR in comparison with Mouse Inoculation Test (MIT) and Fluorescent Antibody Test (FAT)

2019 ◽  
Vol 3 (1) ◽  
pp. 80-85
Author(s):  
Thangaraj Sekar ◽  
Ananda Arone Premkumar ◽  
Ganesan Chandra Mohan ◽  
Balaraman Sekar ◽  
Bheeman Sundaran ◽  
...  
Keyword(s):  
Real Time ◽  
Rabies Virus ◽  
Real Time Pcr ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Inoculation Test

scholarly journals Clarifying Indeterminate Results on the Rabies Direct Fluorescent Antibody Test Using Real-Time Reverse Transcriptase Polymerase Chain Reaction

2018 ◽  
Vol 134 (1) ◽  
pp. 57-62 ◽  
Author(s):  
Kim Appler ◽  
Scott Brunt ◽  
Jodie A. Jarvis ◽  
April D. Davis
Keyword(s):  
Real Time ◽  
Rabies Virus ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
The United States ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Virus Variant ◽  
Direct Fluorescent Antibody ◽  
Polymerase Chain

Objectives: Each year, rabies virus infection results in the death of more than 50 000 persons worldwide. In the United States, the Centers for Disease Control and Prevention (CDC) reported 23 human rabies cases from May 1, 2008, through October 1, 2017. Although rabies testing in the United States is highly reliable, some specimens submitted to rabies laboratories do not have adequate tissues or may be substantially decomposed. In these instances, the specimen may be considered unsatisfactory for testing or produce indeterminate results using the gold standard direct fluorescent antibody test. The objective of this study was to evaluate the number of unsatisfactory samples or samples with indeterminate results that were positive for rabies virus after additional testing using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Methods: In 2016, we retested all unsatisfactory specimens or specimens with indeterminate results using real-time RT-PCR. We further typed any sample that was real-time RT-PCR positive to identify the infecting rabies virus variant. Results: Of 210 retested unsatisfactory specimens or specimens with indeterminate results, 9 (4.3%) were positive for rabies. In each case, the animal was infected with a homologous rabies virus variant. Conclusion: These results confirm the recommendation by CDC and state public health laboratories that indeterminate results should be considered positive and justify the prompt treatment of exposed persons through an animal that is suspected to have rabies.

scholarly journals Real-Time RT-PCR for the Detection of Lyssavirus Species

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
A. Deubelbeiss ◽  
M.-L. Zahno ◽  
M. Zanoni ◽  
D. Bruegger ◽  
R. Zanoni
Keyword(s):  
Real Time ◽  
Rabies Virus ◽  
Fluorescent Antibody ◽  
Fatal Outcome ◽  
Antibody Test ◽  
Rt Pcr ◽  
Causative Agents ◽  
Rabies Virus Strain ◽  
Rabies Diagnosis

The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

Isolation and identification of Marek’s disease virus (MDV) from feather follicle epithelium and internal organs of diseased chickens in Dakahlia Governorate, Egypt

2019 ◽  
Vol 20 (2) ◽  
pp. 6-11
Author(s):  
Aly El-Kenawy ◽  
Mohamed El-Tholoth ◽  
Emad A
Keyword(s):  
Real Time ◽  
Disease Virus ◽  
Real Time Pcr ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Field Samples ◽  
Feather Follicle ◽  
Positive Results

In the present study, a total of 16 samples including feather follicle epithelium, ovary, spleen and kidney (4 samples for each organ) were collected from diseased chicken flocks suspected to be infected with Marek’s disease virus (MDV) at Dakahlia Governorate, Egypt during the period from October 2016 to October 2017. Each sample was pooled randomly from three to five birds (90 to 360 days old). The isolation of the suspected virus from the collected samples was carried out via chorioallantoic membranes (CAMs) of 12 days old embryonated chicken eggs (ECEs). Three egg passages were carried out for each sample. Hyperimmune serum was prepared against standard MDV. MDV in both field and egg passaged samples (after 3rd passage) was identified by agar gel precipitation test (AGPT) and indirect fluorescence antibody test (IFAT). Molecular identification of virus was carried out by conventional polymerase chain reaction (PCR) and real- time PCR in four selected samples. The results revealed that 14 samples (87.5%) including 4 (100%) samples from feather follicle epithelium, ovary and kidney and 2 (50%) samples from spleen, showed positive results in virus isolation after 3rd passage. The positive results percentage by AGPT for field samples were 50% (8 out of 16 samples), while after the 3rd passage in ECEs were 37.5% (6 out of 16 samples) and the positive results percentage by IFAT for field samples were 62.5% (10 out of 16 samples), while after the 3rd passage in ECEs were 81.25 % (13 out of 16 samples). Viral nucleic acid was detected in all selected samples by conventional and real- time PCR. The results indicate that feather follicle epithelium is the best organ for MDV detection. IFAT is superior over AGPT in virus detection. Conventional and real - time PCR could be efficiently used for molecular detection of the virus.

scholarly journals Probe-Based Real-Time qPCR Assays for a Reliable Differentiation of Capripox Virus Species

2021 ◽  
Vol 9 (4) ◽  
pp. 765
Author(s):  
Janika Wolff ◽  
Martin Beer ◽  
Bernd Hoffmann
Keyword(s):  
Real Time ◽  
Animal Health ◽  
Diagnostic Methods ◽  
Virus Species ◽  
Time Saving ◽  
Robust Detection ◽  
Highly Sensitive ◽  
Reliable Differentiation ◽  
World Organization ◽  
Real Time Qpcr

Outbreaks of the three capripox virus species, namely lumpy skin disease virus, sheeppox virus, and goatpox virus, severely affect animal health and both national and international economies. Therefore, the World Organization for Animal Health (OIE) classified them as notifiable diseases. Until now, discrimination of capripox virus species was possible by using different conventional PCR protocols. However, more sophisticated probe-based real-time qPCR systems addressing this issue are, to our knowledge, still missing. In the present study, we developed several duplex qPCR assays consisting of different types of fluorescence-labelled probes that are highly sensitive and show a high analytical specificity. Finally, our assays were combined with already published diagnostic methods to a diagnostic workflow that enables time-saving, reliable, and robust detection, differentiation, and characterization of capripox virus isolates.

scholarly journals Detection of Rabies Virus Antigen in Dog Saliva Using a Latex Agglutination Test

2000 ◽  
Vol 38 (8) ◽  
pp. 3098-3099 ◽  
Author(s):  
S. Kasempimolporn ◽  
W. Saengseesom ◽  
B. Lumlertdacha ◽  
V. Sitprija
Keyword(s):  
Rabies Virus ◽  
Gamma Globulin ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Virus Antigen ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
Glass Slide ◽  
Human Rabies ◽  
Latex Particles

Dog bites are responsible for more than 90% of human rabies deaths in Asia. We developed a simple and inexpensive test based on latex agglutination (LA) for rabies virus antigen detection in dog saliva. Rabies virus antigen could be detected by agglutination on a glass slide using latex particles coated with gamma globulin. By evaluation of paired saliva-brain specimens from 238 dogs, the LA test using saliva was 99% specific and 95% sensitive compared to the fluorescent antibody test (FAT) on brain smears. The advantages of the LA test over the standard FAT are that it is comparatively simple and there is no need to kill the animal before examination.

scholarly journals Antigenic characterisation of lyssaviruses in South Africa

2014 ◽  
Vol 81 (1) ◽  
Author(s):  
Ernest Ngoepe ◽  
Christine Fehlner-Gardiner ◽  
Alex Wandeler ◽  
Claude Sabeta
Keyword(s):  
South Africa ◽  
Monoclonal Antibody ◽  
Rabies Virus ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Reservoir Host ◽  
Differential Staining ◽  
Bat Virus ◽  
Antigenic Reactivity ◽  
Mokola Virus

There are at least six Lyssavirus species that have been isolated in Africa, which include classical rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus and Ikoma lyssavirus. In this retrospective study, an analysis of the antigenic reactivity patterns of lyssaviruses in South Africa against a panel of 15 anti-nucleoprotein monoclonal antibodies was undertaken. A total of 624 brain specimens, collected between 2005 and 2009, confirmed as containing lyssavirus antigen by direct fluorescent antibody test, were subjected to antigenic differentiation. The lyssaviruses were differentiated into two species, namely rabies virus (99.5%) and Mokola virus (0.5%). Furthermore, rabies virus was further delineated into two common rabies biotypes in South Africa: canid and mongoose. Initially, it was found that the canid rabies biotype had two reactivity patterns; differential staining was observed with just one monoclonal antibody. This difference was likely to have been an artefact related to sample quality, as passage in cell culture restored staining. Mongoose rabies viruses were more heterogeneous, with seven antigenic reactivity patterns detected. Although Mokola viruses were identified in this study, prevalence and reservoir host species are yet to be established. These data demonstrate the usefulness of monoclonal antibody typing panels in lyssavirus surveillance with reference to emergence of new species or spread of rabies biotypes to new geographic zones.

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