A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test
A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurate...
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| creator | Nazé, Florence Lamoral, Sophie Suin, Vanessa Van Gucht, Steven Kalai, Michael De Craeye, Stéphane Francart, Aurélie |
| description | A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible. |
| doi_str_mv | 10.1155/2014/256175 |
| format | Article |
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Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.</description><identifier>ISSN: 2314-6133</identifier><identifier>EISSN: 2314-6141</identifier><identifier>DOI: 10.1155/2014/256175</identifier><identifier>PMID: 24822188</identifier><language>eng</language><publisher>Cairo, Egypt: Hindawi Puplishing Corporation</publisher><subject>Animals ; Antigens ; Base Sequence ; Bats ; Biomedical research ; Biopsy ; Brain - virology ; Cats ; Chiroptera ; Computer Simulation ; Dogs ; Fluorescent Antibody Technique ; Humans ; Infections ; Laboratories ; Limit of Detection ; Lyssavirus ; Lyssavirus - classification ; Lyssavirus - genetics ; Lyssavirus - isolation & purification ; Methods ; Mice ; Molecular Sequence Data ; Public health ; Rabies ; Rabies virus ; Real-Time Polymerase Chain Reaction - methods ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction - methods ; Rhabdoviridae Infections - diagnosis ; Rhabdoviridae Infections - virology ; RNA, Viral - analysis ; RNA, Viral - genetics ; Sequence Alignment ; Skin ; Urine ; Vesicular stomatitis virus</subject><ispartof>BioMed research international, 2014-01, Vol.2014 (2014), p.1-12</ispartof><rights>Copyright © 2014 Vanessa Suin et al.</rights><rights>Copyright © 2014 Vanessa Suin et al. Vanessa Suin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</rights><rights>Copyright © 2014 Vanessa Suin et al. 2014</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c499t-97baec273ff956970e00874075ce2e34935558686fa0b1b75e106ba3cd45e36e3</citedby><cites>FETCH-LOGICAL-c499t-97baec273ff956970e00874075ce2e34935558686fa0b1b75e106ba3cd45e36e3</cites><orcidid>0000-0002-8150-9372 ; 0000-0001-9798-3477</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009295/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009295/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,315,728,781,785,886,27929,27930,53796,53798</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24822188$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Stijlemans, Benoît</contributor><creatorcontrib>Nazé, Florence</creatorcontrib><creatorcontrib>Lamoral, Sophie</creatorcontrib><creatorcontrib>Suin, Vanessa</creatorcontrib><creatorcontrib>Van Gucht, Steven</creatorcontrib><creatorcontrib>Kalai, Michael</creatorcontrib><creatorcontrib>De Craeye, Stéphane</creatorcontrib><creatorcontrib>Francart, Aurélie</creatorcontrib><title>A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test</title><title>BioMed research international</title><addtitle>Biomed Res Int</addtitle><description>A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. 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Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.</description><subject>Animals</subject><subject>Antigens</subject><subject>Base Sequence</subject><subject>Bats</subject><subject>Biomedical research</subject><subject>Biopsy</subject><subject>Brain - virology</subject><subject>Cats</subject><subject>Chiroptera</subject><subject>Computer Simulation</subject><subject>Dogs</subject><subject>Fluorescent Antibody Technique</subject><subject>Humans</subject><subject>Infections</subject><subject>Laboratories</subject><subject>Limit of Detection</subject><subject>Lyssavirus</subject><subject>Lyssavirus - classification</subject><subject>Lyssavirus - genetics</subject><subject>Lyssavirus - isolation & purification</subject><subject>Methods</subject><subject>Mice</subject><subject>Molecular Sequence Data</subject><subject>Public health</subject><subject>Rabies</subject><subject>Rabies virus</subject><subject>Real-Time Polymerase Chain Reaction - 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Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. 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| subjects | Animals Antigens Base Sequence Bats Biomedical research Biopsy Brain - virology Cats Chiroptera Computer Simulation Dogs Fluorescent Antibody Technique Humans Infections Laboratories Limit of Detection Lyssavirus Lyssavirus - classification Lyssavirus - genetics Lyssavirus - isolation & purification Methods Mice Molecular Sequence Data Public health Rabies Rabies virus Real-Time Polymerase Chain Reaction - methods Reproducibility of Results Reverse Transcriptase Polymerase Chain Reaction - methods Rhabdoviridae Infections - diagnosis Rhabdoviridae Infections - virology RNA, Viral - analysis RNA, Viral - genetics Sequence Alignment Skin Urine Vesicular stomatitis virus |
| title | A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test |
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