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Design and optimization of a novel reverse transcription linear‐after‐the‐exponential PCR for the detection of foot‐and‐mouth disease virus
Author(s) -
Pierce K.E.,
Mistry R.,
Reid S.M.,
Bharya S.,
Dukes J.P.,
Hartshorn C.,
King D.P.,
Wangh L.J.
Publication year - 2010
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2009.04640.x
Subject(s) - reverse transcriptase , complementary dna , foot and mouth disease virus , rna , biology , microbiology and biotechnology , reverse transcription polymerase chain reaction , polymerase chain reaction , dna , rna extraction , real time polymerase chain reaction , virus , amplicon , virology , messenger rna , gene , genetics
Aims: A novel molecular assay for the detection of foot‐and‐mouth disease virus (FMDV) was developed using linear‐after‐the‐exponential polymerase chain reaction (LATE‐PCR). Methods and Results: Pilot experiments using synthetic DNA targets demonstrated the ability of LATE‐PCR to quantify initial target concentration through endpoint detection. A two‐step protocol involving reverse transcription (RT) followed by LATE‐PCR was then used to confirm the ability of the assay to detect FMDV RNA. Finally, RT and LATE‐PCR were combined in a one‐step duplex assay for co‐amplification of an FMDV RNA segment and an internal control comprised of an Armored RNA ® . In that form, each of the excess primers in the reaction mixture hybridize to their respective RNA targets during a short pre‐incubation, then generate cDNA strands during a 3‐min RT step at 60°C, and the resulting cDNA is amplified by LATE‐PCR without intervening sample processing. Conclusions: The RT‐LATE‐PCR assay generates fluorescent signals at endpoint that are proportional to the starting number of RNA targets and can detect a range of sequence variants using a single mismatch‐tolerant probe. Significance and Impact of the Study: In addition to offering improvements over current laboratory‐based molecular diagnostic assays for FMDV, this new assay is compatible with a novel portable (‘point‐of‐care’) device, the BioSeeq ® II, designed for the rapid diagnosis of FMD in the field.