Development of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detecting Feline Coronavirus

Abstract

Feline infectious peritonitis (FIP) is a worldwide fatal disease caused by a mutant feline coronavirus (FCoV). Simple and efficient molecular detection methods are needed. Here, sensitive, specific, rapid, and reliable colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect the ORF1a/1b gene of FCoV from cats with suspected FIP using neutral red as an indicator. Novel LAMP primers were specifically designed based on the gene of interest. The isothermal assay could visually detect FCoV at 58 °C for 50 min. The RT-LAMP assay was highly specific and had no cross-reactivity with other related feline viruses. The detection limit of FCoV detection by RT-LAMP was 20 fg/µL. A blind clinical test (n = 81) of the developed RT-LAMP procedure was in good agreement with the conventional PCR method. In the light of its performance specificity, sensitivity, and easy visualization, this neutral-red-based RT-LAMP approach would be a fruitful alternative molecular diagnostic tool for veterinary inspection of FCoV when combined with nucleotide sequencing or specific PCR to affirm the highly virulent FIP-associated FCoV.

Keywords: feline infectious peritonitis; molecular diagnosis; reverse transcription loop-mediated isothermal amplification.

Figure 1

Localization of RT-LAMP primer set within ORF1a/1b sequence. The green shaded row presents the FCoV genome consisting of the 5′ untranslated region (5′ UTR), ORF1a/1b, spike (S), ORF3a, ORF3b, ORF3c, envelope (E), membrane (M), nucleocapsid (N), ORF7a, ORF7b, and 3′ untranslated region (3′ UTR).

Figure 2

Specificity of RT-LAMP primers for FCoV detection. Different feline viruses, feline cells, and feline buffy coats amplified using the RT-LAMP primers were detected based on NR indicator dye (upper row) and by agarose gel electrophoresis (lower row). The positive control, WSU 79-1146 (AY994055), was pink color and the negative control (NTC) was yellow color. Lane M, 100 bp Ladder DNA Marker III (Yeastern Biotech, New Taipei City, Taiwan); lane 1-7, FIV, FeLV, FPLV, FCV, FHV, feline cell, FCoV effusion fluid sample no. KU72, respectively; lane 8, PTC, positive control (FIPV strain WSU79-1146, AY994055); lane 9, feline buffy coat; lane NTC, negative control.

Figure 3

Comparative sensitivity of RT-LAMP and RT-PCR for FCoV detection. Pink bar shows the sensitivity of RT-LAMP detected by NR-based method confirmed by agarose gel electrophoresis. Blue bar shows the sensitivity of RT-PCR. Lane M, 100 bp Ladder DNA Marker III (Yeastern Biotech, New Taipei City, Taiwan), lane 1-13, amplification products of 10-fold serially diluted FCoV RNA at 2000 ng/µL, 200 ng/µL, 20 ng/µL, 2 ng/µL, 200 pg/µL, 20 pg/µL, 2 pg/µL, 200 fg/µL, 20 fg/µL, 2 fg/µL, 200 ag/µL, 20 ag/µL, and 2 ag/µL, respectively. Lane NTC, negative control.