Reverse transcription-polymerase chain reaction assay for rabies virus detection

Dantas, Joeler Vargas; Kimura, Leda Maria Silva; Ferreira, Milene Silveira; Fialho, Alexandre Madi; Almeida, Marilda Maria Santos; Grégio, C.R.V.; Romijn, Phyllis Catharina; Leite, José Paulo Gagliardi

doi:10.1590/s0102-09352004000300017

Cited by 9 publications

(5 citation statements)

References 8 publications

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“…It has been made possible to establish a definitive diagnosis by RT‐PCR from decomposed samples that may not be suitable for histology or virus isolation. The few studies undertaken to compare results obtained with RT‐PCR with those obtained using FAT and RT‐PCR showed that the RT‐PCR method was as good as the standard methods of antigen detection and virus isolation (Heaton et al., 1997 and Dantas Junior et al., 2004).…”

Section: Discussionmentioning

confidence: 99%

Pathological, Immunological and Molecular Diagnosis of Rabies in Clinically Suspected Animals of Different Species Using Four Detection Techniques in Jordan

Faizee

Hailat

Ababneh

et al. 2011

Transboundary and Emerging Diseases

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A total of 29 brain tissue samples (BTS) were examined for rabies infection by different diagnostic techniques. None of the examined brain tissues were presented as a whole intact brain. Twenty-seven brain tissue samples from various animal species - dog (13 cases), cat (one case), fox (one case), pig (one case), cow (three cases), sheep (two cases), goat (one case), camel (one case), horse (one case) and donkey (three cases) - were provided by the Vaccine and Sera Department/Al-Bashir Central Hospital in Amman/Jordan from July 2009 up to May 2010. All these samples were frozen at -20°C, for a period of time and then fixed in 10% formalin after being tested for rabies virus by fluorescence antibody test (FAT). The results showed that 21 (77.77%) of 27 BTS were positive for rabies by FAT. Seventeen samples (58.62%) of 29 were positive by histopathology, 2 (6.90%) were positive by histopathology, immunohistochemistry (IHC) and of those which were fixed for 24h only, and 21 (72.42%) were positive using RT-PCR assay. Five of 29 BTS had no pathological lesions, 17 had Negri bodies and the remaining had non-suppurative encephalitis and necrosis. Thirteen BTS that were diagnosed positive by FAT were also positive by RT-PCR and histopathology, but negative by IHC. Four BTS that were positive by FAT were negative by histopathology, IHC and RT-PCR. Also, 3 BTS (cases 19, 22, and 25) that were negative by FAT were positive by RT-PCR and negative by IHC. One of these was negative, while two were positive by histopathology. Therefore, definitive diagnosis of rabies under these conditions in Jordan needs one or more other diagnostic tests in addition to FAT. Also, freezing and prolonged formalin fixation of BTS is not suitable for the detection of rabies virus antigen using IHC.

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Section: Discussionmentioning

confidence: 99%

Pathological, Immunological and Molecular Diagnosis of Rabies in Clinically Suspected Animals of Different Species Using Four Detection Techniques in Jordan

Faizee

Hailat

Ababneh

et al. 2011

Transboundary and Emerging Diseases

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“…All bat brains were tested for lyssavirus by using reverse transcription PCR ( 8 ) and for lyssavirus antigens by using a direct fluorescence antibody test ( 9 ) using fluorescein isothiocyanate–conjugated monoclonal antibodies (Fujirebio Diagnostic, Inc, Malvern, PA, USA). The mouse inoculation test was conducted by using brains of 13 bats that died during capture and transport ( 9 ).…”

mentioning

confidence: 99%

Bat Lyssaviruses, Northern Vietnam

Nguyen¹,

Nguyen²,

Noguchi³

et al. 2014

Emerg. Infect. Dis.

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“…The currently used Gold standard methods used for rabies diagnosis, the Fluorescence Antibody Test (FAT) and the Mouse Inoculation Test (MIT) are time consuming and are highly variable in specificity and sensitivity in the quantification assays. In this connection, the use of the RT-PCR for rabies virus detection was proposed [9] which proved to be a suitable technique for rabies diagnosis by various researches [10][11][12]. RT-PCR assays are used for the detection of Lyssavirus RNA from the samples like saliva, nuchal skin biopsy, eye washes etc with low viral load [13].…”

Section: Issn: 2638-1966mentioning

confidence: 99%

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

Sekar¹,

Premkumar²,

Mohan³

et al. 2019

Madridge J Vaccines

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Real time PCR is the modern molecular techniques used for detection and quantification of viral particle in the specimens. In this study Real Time PCR (RT-PCR) based method was developed and used to quantify the rabies virus PV-11 strains in the different viral harvests of tissue culture fluid in comparison with the Mouse Inoculation Test (MIT) and Fluorescent Antibody Test (FAT). The Eagle's MEM tissue culture medium with 5% fetal calf serum, 1% neomycin and 0.5% Amphotericin-B were used for the propagation of Vero cells in 150 cm 2 tissue culture flasks. The confluent monolayers of the Vero cells (90%) were infected with 0.2 MOI of PV-11 strain of rabies virus. Five individual viral harvests were collected from the infected vero cells at every 3 days interval and subjected for MIT, FAT to quantify the viral titre. The MIT titrated sample was used for the preparation of five point internal reference standard for RT-PCR technique to quantify the number of viral particles in the harvested fluid. The primers targeting 98 base pairs from 446 to 543 in the cDNA of the rabies virus nucleoprotein gene (N gene) were designed and synthesized for Real time PCR assay. The in-house RT-PCR assay was developed with SYBR green master mix. Viral particle in each harvests were quantified in comparison with internal reference standard included in the assay. The results of MIT, FAT and Real time PCR methods were compared with each other. The standardized Real time PCR method was applied for the quantification of viral titre in the different viral harvests cultured using Roller bottle and Bioreactor culturing methods using two different medium. Maximum log of 7.216 viruses per 1 ml of bioreactor viral harvest was quantified by using the real time PCR method. Dilution of the samples are required if the sample having higher viral titre which is not covered within the linear standard range of log 4.50 to log 6.50. This real time PCR based quantification of rabies virus has the advantage of greater accuracy, rapid, economical when compared to the other tests and no animal models required.

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Reverse transcription-polymerase chain reaction assay for rabies virus detection

Abstract: [Protocolo de reação em cadeia pela polimerase precedida de transcrição reversa para a detecção de vírus da raiva]

Cited by 9 publications

References 8 publications

Pathological, Immunological and Molecular Diagnosis of Rabies in Clinically Suspected Animals of Different Species Using Four Detection Techniques in Jordan

Pathological, Immunological and Molecular Diagnosis of Rabies in Clinically Suspected Animals of Different Species Using Four Detection Techniques in Jordan

Bat Lyssaviruses, Northern Vietnam

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

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