Development of TaqMan Probe-Based Insulated Isothermal PCR (iiPCR) for Sensitive and Specific On-Site Pathogen Detection

Abstract

Insulated isothermal PCR (iiPCR), established on the basis of Ralyeigh-Bénard convection, is a rapid and low-cost platform for nucleic acid amplification. However, the method used for signal detection, namely gel electrophoresis, has limited the application of iiPCR. In this study, TaqMan probe-based iiPCR system was developed to obviate the need of post-amplification processing. This system includes an optical detection module, which was designed and integrated into the iiPCR device to detect fluorescent signals generated by the probe. TaqMan probe-iiPCR assays targeting white spot syndrome virus (WSSV) and infectious myonecrosis virus were developed for preliminary evaluation of this system. Significant elevation of fluorescent signals was detected consistently among positive iiPCR reactions in both assays, correlating with amplicon detection by gel electrophoresis analysis. After condition optimization, a threshold value of S/N (fluorescent intensityafter/fluorescent intensitybefore) for positive reactions was defined for WSSV TaqMan probe-iiPCR on the basis of 20 blank reactions. WSSV TaqMan probe-iiPCR generated positive S/Ns from as low as 101 copies of standard DNA and lightly infected Litopenaeus vannamei. Compared with an OIE-certified nested PCR, WSSV TaqMan probe-iiPCR showed a sensitivity of 100% and a specificity of 96.67% in 120 WSSV-free or lightly infected shrimp samples. Generating positive signals specifically and sensitively, TaqMan probe-iiPCR system has a potential as a low-cost and rapid on-site diagnostics method.

A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer

“…The pan-DENV RTiiPCR was designed on the basis of the previously described probe hydrolysis-based POCKIT method (21). The primers and probe targeted a highly conserved region in the 3= UTR of the DENV genome (see Table S1 in the supplemental material).…”

“…Recently, a fluorescent probe hydrolysis-based insulated isothermal PCR (iiPCR) for amplification and detection of nucleic acid has been described (21). The iiPCR is highly sensitive and specific for the detection of both DNA and RNA and could be performed with a single heating source; thus, it does not require an expensive thermocycler (20,21).…”

“…The iiPCR is highly sensitive and specific for the detection of both DNA and RNA and could be performed with a single heating source; thus, it does not require an expensive thermocycler (20,21). The PCR mix in a capillary tube (R-tube; GeneReach USA, Lexington, MA, USA) is heated at the bottom.…”

“…Rayleigh-Bénard convection drives fluid cycling through temperature gradients, and the three PCR steps, namely, denaturation, annealing, and extension, can be completed at different zones within the capillary tube. Subsequent integration of hydrolysis probe technology and an optical detection module into the device allows automatic detection and interpretation of iiPCR results (21). Taking advantage of the fluorescent probe-based iiPCR methodology and a now commercially available reverse transcription-iiPCR (RT-iiPCR) instrument, the POCKIT nucleic acid analyzer (GeneReach USA, Lexington, MA, USA), we developed an RT-iiPCR assay for the detection of DENV RNA of all serotypes in clinical specimens.…”

Rapid and sensitive detection ofFeline immunodeficiency virususing an insulated isothermal PCR-based assay with a point-of-need PCR detection platform

“…according to the default signal-to-noise (S/N) thresholds. 4,17 To determine analytical sensitivity of the established assay, a synthetic plasmid containing the T7 promoter-LTR/ gag region of the FIV genome cassette e was used to prepare RNA transcribed in vitro (IVT) RNA using a commercial kit.…”