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. 2015 Aug:69:16-21.
doi: 10.1016/j.jcv.201505004. Epub 2015 May 19.

Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus

Affiliations

Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus

Ahmed Abd El Wahed et al. J Clin Virol. 2015 Aug.

Abstract

Background: In developing countries, equipment necessary for diagnosis is only available in few central laboratories, which are less accessible and of limited capacity to test large numbers of incoming samples. Moreover, the transport conditions of samples are inadequate, therefore leading to unreliable results.

Objectives: The development of a rapid, inexpensive, and simple test would allow mobile detection of viruses.

Study design: A suitcase laboratory "Diagnostics-in-a-Suitcase" (56c×ばつ45.5c×ばつ26.5cm) containing all reagents and devices necessary for performing a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed. As an example, two RT-RPA assays were established for the detection of hemagglutinin (H) and neuraminidase (N) genes of the novel avian influenza (H7N9) virus.

Results: The sensitivities of the H7 and the N9 RT-RPA assays were 10 and 100 RNA molecules, respectively. The assays were performed at a single temperature (42°C). The results were obtained within 2-7min. The H7N9 RT-RPA assays did not show a cross-detection either of any other respiratory viruses affecting humans and/or birds or of the human or chicken genomes. All reagents were used, stored, and transported at ambient temperature, that is, cold chain independent. In addition, the Diagnostics-in-a-Suitcase was operated by a solar-powered battery.

Conclusions: The developed assay protocol and mobile setup performed well. Moreover, it can be easily implemented to perform diagnoses at airports, quarantine stations, or farms for rapid on-site viral nucleic acid detection.

Keywords: Avian influenza A (H7N9); Diagnostics-in-a-Suitcase; Recombinase polymerase amplification assay.

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Figures

Fig. 1
Fig. 1
Diagnostics-in-a-Suitcase. It contains all equipment and reagents for performing the RT-RPA assay.
Fig. 2
Fig. 2
Layout of the PVC layer of the Diagnostics-in-a-Suitcase. All measurements are in centimeters. The inside of the bold line represents the cutouts whereas the dotted lines represent the distance between two points.
Fig. 3
Fig. 3
Analytical sensitivity of H7N9 RT-RPA assays. (A) The H7 and (B) the N9 RT-RPA assays. Fluorescence development via real-time detection using a dilution range of 107–101 RNA molecules/μl of the H7 and N9 RNA molecular standards (graph generated by ESEquant tube scanner studio software). The sensitivities were 10 and 100 RNA molecules for the H7 (A) and the N9 (B) RT-RPA assays, respectively. The reverse transcription took place in the first minute. To increase the sensitivity, a mixing step was performed after 3 min (no fluorescence signal was detected). 107 represented by black line; 106, gray; 105, red; 104, blue; 103, green; 102, cyan; 101, dark khaki; and negative control, orange. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)
Fig. 4
Fig. 4
Reproducibility of H7N9 RT-RPA assays. (A) H7 and (B) N9 RT-RPA assays. Semilogarithmic regression of the data collected from eight H7N9 RT-RPA test runs on the RNA molecular standard using Prism Software. Both assays yielded results between 2 and 7 min. In the H7 RT-RPA assay, 107–102 RNA molecules were detected in eight out of eight, and 10 in six of eight RT-RPA runs. In the N9 assay, 107–103 RNA molecules were detected in all RT-RPA runs, 102 in seven out of eight, and 10 in two out of eight.
Fig. 5
Fig. 5
Probit analysis of H7N9 RT-RPA assays. (A) H7 and (B) N9 RT-RPA assays. Probit regression analysis using STATISTICA software on the data set of the eight RT-RPA assay runs. The limit of detection at 95% probability (14 and 179 RNA molecules for the H7 and N9 RT-RPA assays, respectively) is represented by a triangle.

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