Design and optimization of a novel reverse transcription linear‐after‐the‐exponential PCR for the detection of foot‐and‐mouth disease virus

Abstract

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.

Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis

“…LATE-PCR is a form of non-symmetric PCR that generates single-stranded DNA making it possible to probe and quantify these strands at endpoint [33–41]. Utilizing both the expanded temperature and fluorescent space now available for detection of targets at endpoint, LATE-PCR assays can readily be multiplexed [40].…”

Rapid Detection and Identification of Hepatitis C Virus (HCV) Sequences using Mismatch-Tolerant Hybridization Probes: A General Method for Analysis of Sequence Variation

“…In contrast, conventional real time PCR cannot take advantage of low temperature detection or mismatched probes because the amplification products become double stranded and prevent probes from hybridizing, which becomes more problematic for longer products (14). The RT-LATE-PCR protocol used here also takes advantage of previously published improvements of pre-incubating primers and targets, short RT incubation at high temperatures, and the use of Tfi polymerase for improved one-step results (11, 15). …”

“…Optimized RT-PCR of HCV AR was carried out as previously described (11) with slight modification. Diluted AR was mixed with limiting and excess primers at concentrations of 250 nM and 5 mM, respectively, in 0.4 mM MgCl 2 , 10 mM TRIS, pH 8.3, and heated to 75C for 3 min to denature capsid proteins, then incubated at ambient temperature for 10 min.…”

Enhanced Reliability and Accuracy for Field Deployable Bioforensic Detection and Discrimination of Xylella fastidiosa subsp. pauca, Causal Agent of Citrus Variegated Chlorosis Using Razor Ex Technology and TaqMan Quantitative PCR

“…The Smart Cycler also has been applied in the identification of Phytophthora ramorum , which causes sudden oak death [21], and the Aphthovirus that causes foot-and-mouth disease [22]. The Razor Ex was designed originally to allow first responders and front line military operations to identify biological threat organisms on-site.…”