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. 2024 Mar 22;19(3):e0297796.
doi: 10.1371/journal.pone.0297796. eCollection 2024.

Development and validation of multiplex one-step qPCR/RT-qPCR assays for simultaneous detection of SARS-CoV-2 and pathogens associated with feline respiratory disease complex

Côme J Thieulent  1   2 , Mariano Carossino  1   2 , Laura Peak  1 , Wendy Wolfson  3 , Udeni B R Balasuriya  1   2
Affiliations

Development and validation of multiplex one-step qPCR/RT-qPCR assays for simultaneous detection of SARS-CoV-2 and pathogens associated with feline respiratory disease complex

Côme J Thieulent et al. PLoS One. .

Abstract

Feline respiratory disease complex (FRDC) is caused by a wide range of viral and bacterial pathogens. Both Influenza A virus (IAV) and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) also induce respiratory diseases in cats. Two one-step multiplex qPCR/RT-qPCR assays were developed and validated: FRA_1 (Feline respiratory assay 1) for the detection of four viral targets and FRA_2 for the detection of three bacteria associated with FRDC. Both multiplex assays demonstrated high specificity, efficiency (93.51%-107.8%), linearity (> 0.998), analytical sensitivity (≤ 15 genome copies/μl), repeatability (coefficient of variation [CV] < 5%), and reproducibility (CV < 6%). Among the 63 clinical specimens collected from FRDC-suspected cats, 92.1% were positive for at least one pathogen and co-infection was detected in 57.1% of samples. Mycoplasma felis (61.9%) was the most found pathogen, followed by feline herpesvirus-1 (30.2%), Chlamydia felis (28.7%) and feline calicivirus (27.0%). SARS-CoV-2 was detected in two specimens. In summary, this new panel of qPCR/RT-qPCR assays constitutes a useful and reliable tool for the rapid detection of SARS-CoV-2 and viral and bacterial pathogens associated with FRDC in cats.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Analytical parameters of singleplex and multiplex qPCR/RT-qPCR assays for the detection of FRDC-associated pathogens and SARS-CoV-2.
A) Comparison of analytical sensitivity of each singleplex and multiplex qPCR/RT-qPCR assays for the detection of pathogens associated with FRDC. B) Analytical sensitivity determination of singleplex and multiplex qPCR/RT-qPCR assays. Each assay was performed using 12 replicates ranging from 103 to 100 copies/μl of IVT DNA/RNA. Each circle and square indicate the Ct value of one replicate obtained by singleplex and multiplex amplification, respectively. Short solid lines indicate the median Ct value and dashed lines indicate the detection limit. Ct: Cycle threshold; IVT RNA: in vitro transcribed RNA; R2: linearity; E: Efficiency; ND: not detected; NTC: no template control.
Fig 2
Fig 2. UpSet plot summarizing the number of feline respiratory pathogens and SARS-CoV-2 detected in felids using the newly developed multiplex qPCR/RT-qPCR panel.
The number of samples with single agents detected or with multiple agents detected (co-infection) are shown as vertical bars. The bottom left horizontal bar graph labeled Set Size shows the total number of samples positives for each specific feline respiratory pathogen and SARS-CoV-2.
See this image and copyright information in PMC

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Grants and funding

This study entitled “Development of multiplex real-time PCR assays for differentiating SARS-CoV-2 from other respiratory and enteric pathogens and enteric pathogens and an IgM/IgG ELISA for the serologic diagnosis of COVID-19 in dogs and cats” was funded to UBRB by the Vet-LIRN COVID-19 Capacity Grant number 1U18FD007514 and supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services (HHS) as part of a financial assistance award totaling $100,000 with 100 percent funding by FDA/HHS. The contents are those of the author(s) and do not necessarily represent the official views of, nor an endorsement, by FDA/HHS, or the U.S. Government. Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration. CJT is partially supported through an NIH-USDA NIFA R01 Research Grant Program Dual Purpose with Dual Benefit: Research in Biomedicine and Agriculture Using Agriculturally Important Domestic Animal Species grant number 2019-67016-29102 (award number AWD-47990-1) from the USDA National Institute of Food and Agriculture to UBRB. Additionally, self-generated funds from UBRB and MC (PG008671) partially supported this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.