Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection

Abstract

Feline infectious peritonitis (FIP), a devastating disease with near-complete mortality, is caused by the feline coronavirus (FCoV) and affects domestic cats worldwide. Herein, we report the development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay incorporating xylenol orange (XO) as a visual indicator for FCoV detection. The assay employed six oligonucleotide primers targeting regions of the nucleocapsid (N) gene. Under optimized conditions (65 °C, 60 min), amplification products were detected through pH-dependent colour changes in the XO dye. The RT-LAMP-XO assay exhibited high specificity for FCoV, with no cross-reactivity against other common feline viral pathogens. While the detection limit (1.7 × 10¹ copies/µL) was an order of magnitude higher than that of qPCR, the method offered advantages in simplicity and speed compared to existing diagnostic approaches. Although less sensitive than qPCR, the RT-LAMP-XO assay may serve as a rapid screening tool when used in combination with additional primer sets. These findings demonstrate the potential utility of XO-based RT-LAMP as a simple, visual detection method for FCoV infection.

Keywords: FIP; RT-LAMP; Xylenol orange; feline corona virus; qPCR.

Figure 1

Schematic diagrams of the FCoV genome comprising the 5’ untranslated region (5’ UTR), open reading frames ORF1a/1b, spike (S) gene, ORF3abc, envelope (E), membrane (M), nucleocapsid (N), ORF7ab, and 3’ untranslated region (3’ UTR).

Figure 2

FCoV nucleotide sequences used for designing LAMP primer. Purple stripes indicate the position of NF3/NB3 primers. Blue stripes indicate the position of NF2/NB2 primers. Green stripes indicate NLF/NLB primers. Orange stripes indicate NF1C/NB1C primers.

Figure 3

FCoV LAMP primer validation. Yellow colour shows positive reaction and violet colour shows negative reaction. NTC, negative control.

Figure 4

Optimization of the colorimetric RT-LAMP-XO assay for FCoV detection involved evaluating several key parameters: (A) the effect of temperature; (B) the effect of incubation time, with the optimal amplification time determined to be 60 min; (C) the effect of MgCl₂ concentration, with 6 mM identified as optimal; (D) the effect of dNTP concentration, with 1.4 mM being optimal; and (E) the effect of Bst DNA/RNA polymerase activity, with 0.32 U/µL selected as the optimum enzyme activity. Lane M represents the 100 bp DNA Ladder Marker III (Yeastern Biotech, Taiwan, China), and lane NTC is the negative control using RNase-free water.

Figure 5

Specificity of the RT-LAMP-XO assay. (A) Colorimetric specificity of RT-LAMP assay using xylenol orange indicator dye. (B) Fluorescence signal results of the specificity analysis. FCoV, feline coronavirus; FCV, feline calicivirus; FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; FPV, feline panleukopenia virus; CRFK, Crandell-Rees Feline Kidney; NTC, Negative control.

Figure 6

Similarity analysis of the FCoV clinical sample sequence.

Figure 7

Comparison of the sensitivity of RT-LAMP-XO and qPCR for FCoV detection. (A) Colorimetric RT-LAMP-XO reaction. Tube a–g: RT-LAMP-XO amplicon of 10-fold serially diluted standard plasmids pGEMT-N at 1.7 × 10⁶ copies/µL to 1.7 × 10¹ copies/µL. NTC: negative control. (B) Sensitivity of quantitative RT-LAMP-XO. (C) Sensitivity of qPCR assay.