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.

Feline calicivirus (FCV) is a highly contagious virus that causes upper respiratory tract disease, commonly known as cat flu. It is widely distributed worldwide and poses a major threat to feline health. Therefore, it is essential to find an efficient and rapid method for detecting FCV. In this study, the colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, using neutral red as an indicator, was developed and validated to target the ORF2 gene of FCV for the first time. Additionally, the study compared the diagnostic abilities of polymerase chain reaction (PCR), nested PCR, and RT-LAMP assays for detecting FCV in clinical samples. The optimized RT-LAMP amplification was carried out at 56.3 °C. The technique visually detected FCV within 70 min, with a limit of detection of 14.3 × 10¹ copies/µL, and showed no cross-reactivity with other feline pathogens. Out of 54 oropharyngeal swab samples, 17 tested positive for FCV using both nested PCR and RT-LAMP, while only one tested positive using conventional PCR. The positivity rate was higher with nested PCR and RT-LAMP (31.48%) compared to conventional PCR (1.85%). Consequently, these results demonstrated the effectiveness of the colorimetric RT-LAMP assay developed in this study as an alternative for diagnosing FCV in cats.

Background and Aim: Feline immunodeficiency virus (FIV) is a retroviral pathogen globally responsible for immunodeficiency disease in cats. However, the current diagnosis based on antibody detection has limitations and can also produce false-positive results. This study aimed to develop a one-pot loop-mediated isothermal amplification (LAMP) process integrated with neutral red (NR-LAMP) assay for detection of FIV proviral DNA. Materials and Methods: We developed a one-pot, gag gene-based NR-LAMP for convenient, rapid, specific, and sensitive colorimetric inspection of FIV proviral DNA. Results: The developed NR-LAMP was capable of amplifying at an optimum temperature of 65°C for 40 min. No cross-amplification was detected between FIV and other feline viruses tested, indicating the high specificity (98.44%) of the novel FIV-LAMP primer. Our NR-LAMP assay has a detection limit of 4.2 × 101 copies/μL. A total of 80 clinical samples with a background of FIV infection were collected and tested using the proposed method. The NR-LAMP assay showed a high sensitivity of 100% compared to conventional polymerase chain reaction assay. Conclusion: These results support the suitability of NR-LAMP as a potential future alternative clinical molecular approach for further use in the diagnosis of FIV-infected cats. Keywords: feline immunodeficiency virus, loop-mediated isothermal amplification, molecular diagnosis, neutral red.

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.

Methicillin-resistant Staphylococcus epidermidis (MRSE) contamination is commonly found on human skin and medical devices. Herein, we present a sensor utilizing molecularly imprinted polymer nanobodies (nanoMIP) for recognition and electrochemical impedance spectroscopy (EIS) to detect S. epidermidis. Sensor manufacturing involves synthesizing nanoMIP via solid-phase synthesis using whole bacteria as templates. Screen-printed gold electrode (AuSPE)-modified 16-mercaptohexadecanoic acid (MHDA) served to immobilize the nanoMIPs on the sensor surface through an amide bond, with the remaining functional groups blocked by ethanolamine (ETA). Scanning electron microscope (SEM) analysis of the modified AuSPE surface reveals immobilized spherical nanoMIP particles of 114–120 nm diameter, while atomic force microscope (AFM) analysis showed increased roughness and height compared to bare AuSPE. The sensor is selective for S. epidermidis, with a remarkable detection limit of 1 CFU/mL. This research demonstrates that the developed nanoMIP-based sensor effectively detects S. epidermidis. Further research will focus on developing protocols to integrate the nanoMIP-based EIS sensor into medical and industrial applications, ultimately contributing to improved safety for both humans and animals in the future.

This study was conducted to identify the molecular and biological characteristics of the collected feline coronavirus (FCoV) strains in northern Vietnam. A total of 166 fecal samples were collected from domestic cats in Hanoi, Bacgiang, Hungyen, and Hanam in northern Vietnam. Conventional polymerase chain reaction (PCR) was used to detect the FCoV genome in the field samples. Results indicated that 19 (11.45%) out of 166 fecal samples were positive for the FCoV genome using PCR. Insignificant differences in positive rates were detected according to breed, age, sex, and health status of the cats. By using nested PCR, all 19 FCoV strains obtained belonged to type I. Analysis of the partial S gene sequences indicated that the nine Vietnamese FCoV strains in this study shared high nucleotide identity, ranging from 88.5% to 99.16%. Phylogenetic analysis supported that the Vietnamese FCoV strains obtained were type I viruses, which are divided into several clades. The nine Vietnamese FCoV strains were genetically close to viruses from the Netherlands, France, and China and differed from the vaccine strain. No mutation was detected among the nine Vietnamese FCoV strains at sites 23,531 and 23,537 in the S gene sequences. The S1/S2 cleavage site on the S protein of FCoV of the nine viral strains indicated an R/G-R-S/A-R-R-S motif, which was typical for feline enteric coronavirus strains. The sequences found were consistent with FECV/low-path viruses, and that it was healthy cats (not FIP cats) that were sampled.