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. 2022 Sep 7;14(9):1982.
doi: 10.3390/v14091982.

A Naked-Eye Visual Reverse Transcription Loop-Mediated Isothermal Amplification with Sharp Color Changes for Potential Pen-Side Test of Foot-and-Mouth Disease Virus

Jie Zhang  1   2 , Qian Hou  2 , Weimin Ma  2 , Danian Chen  2 , Weibing Zhang  2 , Ashenafi Kiros Wubshet  2 , Yaozhong Ding  2 , Miaomiao Li  2 , Qian Li  2 , Jiao Chen  2 , Junfei Dai  2 , Guohua Wu  2 , Ziteng Zhang  1 , Alexei D Zaberezhny  3 , Zygmunt Pejsak  4 , Kazimierz Tarasiuk  4 , Muhammad Umar Zafar Khan  5 , Yang Wang  2 , Jijun He  2 , Yongsheng Liu  1
Affiliations

A Naked-Eye Visual Reverse Transcription Loop-Mediated Isothermal Amplification with Sharp Color Changes for Potential Pen-Side Test of Foot-and-Mouth Disease Virus

Jie Zhang et al. Viruses. .

Abstract

Visual loop-mediated isothermal amplification (LAMP) is qualified to be applied in the field to detect pathogens due to its simplicity, rapidity and cost saving. However, the color changes in currently reported visual reverse transcription LAMP (RT-LAMP) for foot-and-mouth disease virus (FMDV) detection are not so obvious to the naked eye, so interpretation of results is troublesome. In this study, a new naked-eye visual RT-LAMP to detect all seven distinct serotypes of FMDV was established based on the 3D genes by using pH-sensitive neutral red as the indicator, rendering a sharp contrast of color changes between the negative (light orange) and the positive (pink). Analytical sensitivity tests showed that the detection limit of the visual RT-LAMP was 104 copies/µL while those were 103 and 104 copies/µL for the RT-qPCR and conventional RT-PCR methods, respectively. Specificity tests proved that the established visual RT-LAMP assay had no cross-reactivity with other common livestock viruses. Furthermore, the analysis of 59 clinical samples showed 98.31% and 100% concordance with the RT-qPCR and the RT-PCR, respectively. The pan-serotypic FMD visual RT-LAMP assay could be suitable for a pen-side test of all seven serotypes of FMDV because the results could be easily distinguished by the naked eye without the requirement of complicated instruments and professional technicians. Hence, the novel method may have a promising prospect in field tests which exert an important role in monitoring, preventing, and controlling FMD, especially in regions with no PCR or qPCR instrument available.

Keywords: 3D gene; foot-and-mouth disease virus; loop-mediated isothermal amplification; naked-eye visualization; pen-side test.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The selection of the optimal reaction conditions of the visual RT-LAMP assay. (A) The selection of the visual RT-LAMP primer sets measured by the fluorescent curves. Totally 6 sets of candidate LAMP primers were tested. Different primer sets were represented in different colors. (B) The selection of the optimal incubation temperatures measured by the fluorescent curves. The fluorescence signals under different temperatures were represented in different colors. (C) The selection of the optimal incubation temperatures also measured by electrophoresis. Lane M, DL2000 DNA marker (Takara, Dalian, China); Lane 1–8, different incubation temperatures subjected to the visual RT-LAMP assay (1, 59 °C; 2, 60 °C; 3, 61 °C; 4, 62 °C; 5, 63 °C; 6, 64 °C; 7, 65 °C; 8, 66 °C); Lane 9, positive control; Lane 10, negative control. (D) The selection of the optimal incubation time was measured by electrophoresis. The electrophoretic results of the visual RT-LAMP products were generated by incubation for a different time at 61 °C. Lane M, DL2000 DNA marker (Takara, Dalian, China); Lane 1–8, different incubation time subjected to the visual RT-LAMP assay (1, 30 min; 2, 40 min; 3, 45 min; 4, 50 min; 5, 55 min; 6, 60 min; 7, 65 min; 8, 70 min); Lane 9, negative control; Lane 10, positive control.
Figure 2
Figure 2
Target region of the visual RT-LAMP primers for pan-serotypic FMDV detection is shown, and reference sequence is KC440882 for FMDV type A isolate EGY 1/2012.
Figure 3
Figure 3
The selection of the indicator dye for the naked-eye visual RT-LAMP assay. The comparison of the color change between the positive and negative reactions of the assay with six different indicator dyes including HNB, calcein, SYBR green I, neutral red, cresol red, and BTB. The results were presented in both white and black backgrounds with positive and negative reactions. Positive reactions were shown by “+” and negative reactions were shown by “−”.
Figure 4
Figure 4
The analytical specificity of the naked-eye visual RT-LAMP assay. (A) Electrophoresis of self-identification of the viruses for cross-reaction analysis by the specific PCR or RT-PCR. Lane 1–10 were ORFV (1137 bp), SPPV (600 bp), PRV (356 bp), PCV2 (250 bp), BVDV (300 bp), SVV (800 bp), CSFV (424 bp), BTV (1200 bp), PRRSV (245 bp, 335 bp) and PPV (445 bp). Lane M, DL2000 DNA marker (Takara, Dalian, China). The identified viruses above were subject to the naked-eye visual RT-LAMP assay for FMDV detection. The amplified products were shown by agarose gel electrophoresis (B) and naked-eye examination in ambient light (C), respectively. Among them, No. 1–11 represented FMDV, ORFV, SPPV, PRV, PCV2, BVDV, SVV, CSFV, BTV, PRRSV, and PPV, respectively. No. 12–13 represented cell culture supernatant and ddH2O as the negative controls. M: DL2000 DNA marker (Takara, Dalian, China).
Figure 5
Figure 5
The comparison of analytical sensitivity of the visual naked-eye RT-LAMP assay between the RT-qPCR and conventional RT-PCR. LOD of the naked-eye visual RT-LAMP (A), the RT-qPCR (B), and the RT-PCR (C) assays for the amplification of FMDV 3D gene RNA transcript standards. Tubes, lines, and lanes 1–8 represent ten-fold serial dilutions (from 107 to 100 copies/µL) of the RNA transcripts. Tube 9 and 10 represent positive and negative control, respectively.
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