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

Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3′ untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n = 220) and individuals not suspected of dengue virus infection (n = 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries

Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer

International audienceEquine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48-99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens

'Frågor'

<div><p>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 intensity_after/fluorescent intensity_before) 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 <em>Litopenaeus vannamei</em>. 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.</p> </div

Time course of WSSV TaqMan probe-iiPCR.

<p>WSSV TaqMan probe-iiPCR reactions containing 10¹ and 10³ copies of pWSSV1 were allowed to be carried out for 5, 10, 15, 20, 25, or 30 min. Amplicons were detected by 12% polyacrylamide gel analysis (A), and the intensity of the bands was estimated by densitometry (ImageJ program, NCBI) (B). In addition, fluorescent signals of three independent experiments were collected, and S/N ratios and SDs were calculated (C). Arrows, iiPCR amplicons; M, DNA size markers; S/N ratio, fluorescent intensity_after/fluorescent intensity_before.</p

Analytical sensitivity of WSSV TaqMan probe-iiPCR.

<p>Ten-fold serial dilutions (10³ to 0 copies) of plasmid DNA were subjected to WSSV TaqMan probe-iiPCR amplification. Amplicons were detected by 12% polyacrylamide gel analysis (A). Fluorescent signals were collected before and after the reaction, and S/N ratios and SDs were calculated (B). Arrows, iiPCR amplicons; M, DNA size markers; “before”, fluorescent signals detected before reaction; “after”, fluorescent signals detected after reaction; S/N ratio, fluorescent intensity_after/fluorescent intensity_before.</p

Detection of WSSV DNA in shrimp samples by IQ 2000 WSSV D&P System, WSSV TaqMan probe-based iiPCR, and real-time PCR assays.

a<p>Fluorescent signals in TaqMan probe-iiPCR assays were recorded before and after the reaction. S/N ratio =  signal intensity_after/signal intensity_before.</p>b<p>The standard curve was established using serial ten-fold dilutions (10⁴ to 10¹ copies) of standard DNA. R² and slope of the WSSV real-time PCR assay were 0.994 and −3.474, respectively.</p

Generation and detection of TaqMan probe hydrolysis in iiPCR.

<p>Target pWSSV1 and pIMNV plasmids (10³ copies) were subjected to WSSV (A) and IMNV (C) iiPCR, respectively, for 30 min in the presence of WSSVprobe, IMNVprobe, or no probe. Fluorescent signals (520 nm) of individual reactions were collected before and after iiPCR by the modified iiPCR and shown below the gel image. After the reactions were completed, amplicons were also analyzed on a 12% polyacrylamide gel in 1X TAE buffer. WSSV (B) and IMNV (D) real time PCR assays containing the same components as in iiPCR were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045278#s2" target="_blank">Materials and Methods</a>. The result shown here is a representative of at least three experiments with similar results. Arrows, iiPCR amplicons; M, DNA size markers; NTC, no template control (ddH₂O); “before”, fluorescent signals detected before reaction; “after”, fluorescent signals detected after reaction; S/N ratio, signal intensity_after/signal intensity_before for iiPCR; signal intensity_{cycle 40}/signal intensity_{cycle 1} for real time PCR.</p

Effects of concentration of TaqMan probe on production of fluorescent signals.

<p>Different concentrations (9.4, 18.8, 37.5, 75, 150, or 300 nM) of WSSV probe or IMNV probe were added to the WSSV (A) or IMNV (B) iiPCR, respectively. Each reaction was performed in triplicate. Mean S/N ratio of each reaction (shown on each mark) was plotted against probe concentration. Mean S/N ratios for reactions containing no probe (not shown on the plot) were 0.97 (SD = 0.08) and 0.94 (SD = 0.01) for WSSV and IMNV assays, respectively. Bottom panel shows an example of gel electrophoresis analysis. S/N ratio, fluorescent intensity_after/fluorescent intensity_before. Amplicons were analyzed on a 12% polyacrylamide gel in 1X TAE buffer. M, DNA size markers.</p

Agreement of WSSV DNA detection between IQ2000 WSSV D&P System and WSSV TaqMan probe-based iiPCR.

<p>Agreement of WSSV DNA detection between IQ2000 WSSV D&P System and WSSV TaqMan probe-based iiPCR.</p