Multiplex One-Step RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Enteric Viruses of Dogs and Cats

Thieulent, Côme J.; Carossino, Mariano; Peak, Laura; Wolfson, Wendy; Balasuriya, Udeni B. R.

doi:10.3390/v15091890

Cited by 9 publications

(16 citation statements)

References 68 publications

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“…In view of our data, we have demonstrated that using our assays in a multiplex format did not affect the analytical parameters. Additionally, these parameters were similar to those from other assays recently developed in our laboratory for the detection of canine enteric viruses and feline respiratory pathogens [69,70]. In this study, two to three log 10 improvement of the LOD 95 was determined when compared to the previously published multiplex PCR assays for the detection of CAdV-2, CDV, CIV, and CPiV [38].…”

Section: Discussionsupporting

confidence: 67%

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex

Thieulent,

Carossino,

Peak

et al. 2023

Viruses

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Canine infectious respiratory disease complex (CIRDC) is the primary cause of respiratory disease in the canine population and is caused by a wide array of viruses and bacterial pathogens with coinfections being common. Since its recognition in late 2019, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been reported to cause respiratory disease in dogs. Therefore, the rapid detection and differentiation of SARS-CoV-2 from other common viral and bacterial agents is critical from a public health standpoint. Here, we developed and validated a panel of four one-step multiplex qPCR/RT-qPCR assays for the detection and identification of twelve pathogens associated with CIRDC (canine adenovirus-2, canine distemper virus, canine herpesvirus-1, canine influenza A virus, canine parainfluenza virus, canine pneumovirus, canine respiratory coronavirus, SARS-CoV-2, Bordetella bronchiseptica, Streptococcus equi subsp. zooepidemicus, Mycoplasma cynos, and M. canis), as well as the identification of three main CIV subtypes (i.e., H3N2, H3N8, and H1N1). All developed assays demonstrated high specificity and analytical sensitivity. This panel was used to test clinical specimens (n = 76) from CIRDC-suspected dogs. M. canis, M. cynos, and CRCoV were the most frequently identified pathogens (30.3%, 25.0%, and 19.7% of samples, respectively). The newly emerging pathogens CPnV and SARS-CoV-2 were detected in 5.3% of samples and coinfections were identified in 30.3%. This new multiplex qPCR/RT-qPCR panel is the most comprehensive panel developed thus far for identifying CIRDC pathogens, along with SARS-CoV-2.

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Section: Discussionsupporting

confidence: 67%

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex

Thieulent,

Carossino,

Peak

et al. 2023

Viruses

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“…These positive samples for CCoV, CRV, CPV and CDV had Ct values of 7.68- 35.83, 15.34-35.22, 8.46-35.06, and 9.25-35.11, respectively. The positivity rates of CCoV, CRV, CPV, and CDV in the 1028 clinical samples with the reference assay [24] were 9.05% (93/1028), 0.88% (9/1028), 25.68% (264/1028), and 4.86% (50/1028), respectively.…”

Section: Detection Results Of Clinical Samplesmentioning

confidence: 97%

Section: Detection Of Clinical Samplesmentioning

confidence: 99%

“…The 1028 clinical samples collected from December 2021 to June 2023 were tested by multiplex RT-qPCR, as reported by Thieulent et al [24], for CCoV, CPV, CRV, and/or CDV. The assay described by Thieulent et al [24] used quadruplex RT-qPCR to detect the CCoV ORF5 gene, CPV VP2 gene, CDV Phosphoprotein gene, and canine adenovirus-1 (CAdV-1) Hexon gene, and it used duplex RT-qPCR to detect the CRV NSP3 gene and SARS-CoV-2 N gene. The assay had analytical sensitivity (LOD) of 6 copies/µL (150 copies/reaction) for CCoV, 14 copies/µL (350 copies/reaction) for CPV, 5 copies/µL (125 copies/reaction) for CDV, and 35 copies/µL (875 copies/reaction) for CRV.…”

Section: Detection Of Clinical Samplesmentioning

confidence: 99%

“…The assay had analytical sensitivity (LOD) of 6 copies/µL (150 copies/reaction) for CCoV, 14 copies/µL (350 copies/reaction) for CPV, 5 copies/µL (125 copies/reaction) for CDV, and 35 copies/µL (875 copies/reaction) for CRV. In our study, these 1028 clinical samples were tested for CCoV, CPV, CDV using triplex RT-qPCR, and were tested for CRV using singleplex RT-qPCR described in the reference assay [24].…”

Section: Detection Of Clinical Samplesmentioning

confidence: 99%

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A Quadruplex Reverse Transcription Quantitative Polymerase Chain Reaction for Detecting Canine Coronavirus, Canine Rotavirus, Canine Parvovirus, and Canine Distemper Virus

Shi,

Long,

Shi

et al. 2024

Microbiology Research

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Background: Canine coronavirus (CCoV), canine rotavirus (CRV), canine parvovirus (CPV), and canine distemper virus (CDV) cause gastroenteritis in dogs, and co-infections of these pathogens are common in China. In particular, CCoV and CRV are confirmed to have important zoonotic potential and cause public health issues. It is difficult to diagnose these diseases based only on clinical manifestations and pathological damage. Methods: In this study, four pairs of specific primers and probes targeting the CCoV M, CRV VP7, CPV VP2, and CDV N genes were designed. The reaction conditions, including the primer and probe concentrations, annealing temperatures, and reaction cycles, were optimized for the development of a quadruplex RT-qPCR for the detection of CCoV, CRV, CPV, and CDV. The assay was used to test 1028 clinical samples to validate its application. Results: A quadruplex RT-qPCR was successfully established for the differential detection of CCoV, CRV, CPV, and CDV, with good specificity, high sensitivity, and excellent repeatability. The assay could specifically detect CCoV, CRV, CPV, and CDV without cross-reactivity with the other canine viruses tested. It showed high sensitivity with limits of detection (LOD) of 1.1 × 102 copies/reaction for all four plasmid constructs. It showed excellent repeatability, with 0.05–0.90% intra-assay variation and 0.02–0.94% inter-assay variation. The 1028 clinical samples were tested using the quadruplex RT-qPCR and a reported reference RT-qPCR. The positivity rates of CCoV, CRV, CPV, and CDV were 9.53%, 0.97%, 25.68%, and 5.06% using the developed assay, and 9.05%, 0.88%, 25.68%, and 4.86% using the reference assay, with agreements higher than 99.32%. Conclusion: The results indicated that a rapid and accurate quadruplex RT-qPCR was developed for the detection and differentiation of CCoV, CRV, CPV, and CDV.

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Burden of Common Respiratory Pathogens Among Cats in China

Umar,

Muhammad,

mouzahim

et al. 2024

Veterinary Medicine & Sci

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BackgroundFeline respiratory disease complex (FRDC) is a set of illnesses which are primarily associated with different types of viruses and bacteria. There is scarcity of data on pathogens associated with FRDC in China.ObjectivesThe primary objective of this study was to investigate the prevalence and dynamics of FRDC pathogens in China.MethodsA total of 458 samples were retrieved from veterinary clinics during 2021–2024 from cats suffering from respiratory infections. Four viruses and three bacteria associated with FRDC were targeted for molecular detection with real time qPCR/RT‐qPCR assays.ResultsAt least 1 targeted respiratory pathogen was detected in 423 samples (92.3%), whereas no pathogens were detected in 7.7% of samples. Bacteria were detected in 32.1% of samples, and viruses were detected in 60.2% of samples. The prevalence of viruses detected were feline calicivirus (31.2%), feline herpesvirus‐1 (24.6%), influenza A virus (2.8%) and severe acute respiratory syndrome coronavirus‐2 (1.5%), whereas the detection rate for bacteria was Mycoplasma felis (15.5%), Chlamydia felis (10.2%) and Bordetella bronchiseptica (6.3%). Significantly higher cases were reported from kittens (57.4%). Pathogen detection was more common during the cold season. Mono‐infections involving one bacteria or virus were detected in 44.7% of samples, whereas coinfections were detected in 47.5% of samples. No quadruple coinfections were recorded in this study.ConclusionsThe frequency of detection of feline respiratory pathogens alone or in combinations among diseased cats was high, indicating a heavy burden of respiratory infections among cats in Kunshan, China. Continued surveillance is desired, and newly emerged respiratory pathogens should also be monitored in routine diagnostic testing.

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Multiplex One-Step RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Enteric Viruses of Dogs and Cats

Cited by 9 publications

References 68 publications

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex

A Quadruplex Reverse Transcription Quantitative Polymerase Chain Reaction for Detecting Canine Coronavirus, Canine Rotavirus, Canine Parvovirus, and Canine Distemper Virus

Burden of Common Respiratory Pathogens Among Cats in China

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