Detection of Bovine Coronavirus by Conventional Reverse Transcription Polymerase Chain Reaction

Alfieri, Amauri Alcindo; Alfieri, Alice Fernandes; Takiuchi, Elisabete

doi:10.1007/978-1-4939-3414-0_9

Cited by 5 publications

(8 citation statements)

References 13 publications

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“…Sensitivity of the TaqMan qRT-PCR assay was increased by 100-fold as compared to universal PCR with a good inter-assay and intra-assay reproducibility. These outcomes are in agreement with previous studies [1,7]. Additionally, the diagnostic sensitivity and specificity were determined using 312 clinical samples from six cattle herds.…”

Section: Resultssupporting

confidence: 88%

Specific Detection of Bovine Coronavirus N Protein with TaqMan Probe qRT-PCR

Geng

Gong

et al. 2019

Acta Scientiae. Vet.

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Background: Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infection may cause losses to production by reduced weight gain, reduced milk yield. Several methods have been applied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRT-PCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technical assay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene. The main objective of the present study to establish a novel TaqMan probe real-time PCR (qRT-PCR) for detecting BCoV.Materials, Methods & Results: The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR) for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collection and processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from the known sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimized including concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, the recombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collected from diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCR were 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assay could specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL. However, universal PCR could detect only 4.72 × 103 copies/μL. Its sensitivity was 100-fold stronger than universal PCR. In conclusion, this TaqMan qRT-PCR had excellent specificity, sensitivity and stability with a 100-fold sensitivity stronger than universal PCR. Minimum detection limit was 4.72 × 101 copies/μL. This method was a cost-effective method to diagnose diarrhea and distinguish pathogens in dairy farms.Discussion: In this study, the authors developed a quantitative real-time PCR (qRT-PCR) in this study based on the TaqMan probe of BCoV. This TaqMan qRT-PCR assay selected and used one pair of specific primers (BCoV-qF/BCoV-qR) and a specific TaqMan probe (BCoV-probe) targeting the conserved nucleocapsid (N) gene. The specificity of primers and probes was validated with Primer-BLAST. The specificity of the qRT-PCR was confirmed by the negative control and other six viruses. The findings demonstrated that TaqMan qRT-PCR could only detect BCoV. This verified the qRT-PCR had an excellent specificity. It is obvious that this TaqMan qRT-PCR assay can detect only BCoV with stronger sensitivity and reproducibility than other real-time PCR methods. The sensitivity test indicated the minimum detection limit of the TaqMan qRT-PCR was 4.72 × 101copies/μL, or 47.2 copies/μL. Sensitivity of the TaqMan qRT-PCR assay was increased by 100-fold as compared to universal PCR with a good inter-assay and intra-assay reproducibility. Thereby, based on the high sensitivity of the assay of this qRT-PCR assay it may be a cost-effective method to diagnose BCoV infections and indentify the etiologic agents of diarrhea syndrome in the dairy farms.

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Section: Resultssupporting

confidence: 88%

Specific Detection of Bovine Coronavirus N Protein with TaqMan Probe qRT-PCR

Geng

Gong

et al. 2019

Acta Scientiae. Vet.

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“…In terms of detection of BCoV, PCR is the gold standard assay and is the most widely used assay for diagnostic and research purposes [31,40,50]. Traditionally, these PCR tests have been based on single detection with specific primers and markers used to facilitate and enhance the detection of the pathogen [9,10,50,51]. An advantage of all PCR tests is that they provide high levels of sensitivity and specificity [9,10,40,50,51].…”

Section: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

“…Traditionally, these PCR tests have been based on single detection with specific primers and markers used to facilitate and enhance the detection of the pathogen [9,10,50,51]. An advantage of all PCR tests is that they provide high levels of sensitivity and specificity [9,10,40,50,51]. This is provided with the use of well-designed primer sequences specific for a particular target site in the organism's genome to facilitate selective and precise annealing of these nucleotides, followed by amplification and detection.…”

Section: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

“…Thus, assays are typically designed to target highly conserved regions of pathogens. The nucleocapsid (N) gene of BCoV is used as a good target for PCR assays as it is well conserved among strains and thus improves the sensitivity of the assay [10,47,51,54]. Escutenaire and colleagues (2007) made use of the BCoV ORF1b region in their RT-PCR assay, citing this as a more conserved site than the N gene and thus better for detecting viruses from the different clusters [50].…”

Section: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

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Advances in Laboratory Diagnosis of Coronavirus Infections in Cattle

van den Hurk,

Regmi,

Naikare

et al. 2024

Preprint

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Coronaviruses cause infections in humans and diverse species of animals and birds with a global distribution. Bovine coronavirus (BCoV) produces predominantly two forms of disease in cattle, a respiratory form, and a gastrointestinal form. All age groups of cattle are affected by the respiratory form of coronavirus whereas the gastroenteric form causes neonatal diarrhea or calf scour in young cattle and winter dysentery in adult cattle. The tremendous impacts of bovine respiratory disease and the associated losses are well-documented and underscores the importance of this pathogen. Beyond this, studies have demonstrated significant impacts on milk production associated with outbreaks of winter dysentery, with up to a 30% decrease in milk yield. Research data so far have shown that the viral structure or strain is not a determinant of tissue tropism and clinical form of the disease. In North America, BCoV was identified for the first time in 1972, and it continues to be a significant economic concern for the cattle industry. The virus is classified under Coronaviridae family, Betacoronavirus genus and Betacorovirus 1 species. A number of conventional and molecular diagnostic assays are available for the detection of BCoV from clinical samples. Conventional assays for BCoV detection include virus isolation, which is challenging from clinical samples, electron microscopy, fluorescent antibody assays, and various immunoassays. Molecular tests are mainly based on nucleic acid detection and predominantly include conventional and real-time polymerase chain reaction (PCR) assays. Isothermal amplification assays and genome sequencing have also been used increasingly in recent years for the detection and identification of BCoV. Loop-mediated isothermal amplification, helicase-dependent amplification, recombinase polymerase amplification, and multienzyme isothermal rapid amplification are some examples of the isothermal amplification assays for used BCoV. Point-of-care testing has gained some popularity recently as they can offer user-friendly testing on the farm (barn-side). The present study reviewed literature on coronavirus infections in cattle from the last three and a half decades and presents information mainly on the current and advancing diagnostics in addition to epidemiology, clinical presentations, and the impact of the disease on the cattle industry.

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“…In terms of the detection of BCoV, PCR is the gold standard assay and the most widely used assay for diagnostic and research purposes [44,48,49]. Traditionally, these PCR tests have been based on single detection with specific primers and markers used to facilitate and enhance the detection of the pathogen [16,17,47,48]. An advantage of all PCR tests is that they provide high levels of sensitivity and specificity [16,17,[47][48][49].…”

Section: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

Advances in Laboratory Diagnosis of Coronavirus Infections in Cattle

van den Hurk,

Regmi,

Naikare

et al. 2024

Pathogens

View full text Add to dashboard Cite

Coronaviruses cause infections in humans and diverse species of animals and birds with a global distribution. Bovine coronavirus (BCoV) produces predominantly two forms of disease in cattle: a respiratory form and a gastrointestinal form. All age groups of cattle are affected by the respiratory form of coronavirus, whereas the gastroenteric form causes neonatal diarrhea or calf scours in young cattle and winter dysentery in adult cattle. The tremendous impacts of bovine respiratory disease and the associated losses are well-documented and underscore the importance of this pathogen. Beyond this, studies have demonstrated significant impacts on milk production associated with outbreaks of winter dysentery, with up to a 30% decrease in milk yield. In North America, BCoV was identified for the first time in 1972, and it continues to be a significant economic concern for the cattle industry. A number of conventional and molecular diagnostic assays are available for the detection of BCoV from clinical samples. Conventional assays for BCoV detection include virus isolation, which is challenging from clinical samples, electron microscopy, fluorescent antibody assays, and various immunoassays. Molecular tests are mainly based on nucleic acid detection and predominantly include conventional and real-time polymerase chain reaction (PCR) assays. Isothermal amplification assays and genome sequencing have gained increased interest in recent years for the detection, characterization, and identification of BCoV. It is believed that isothermal amplification assays, such as loop-mediated isothermal amplification and recombinase polymerase amplification, among others, could aid the development of barn-side point-of-care tests for BCoV. The present study reviewed the literature on coronavirus infections in cattle from the last three and a half decades and presents information mainly on the current and advancing diagnostics in addition to epidemiology, clinical presentations, and the impact of the disease on the cattle industry.

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Detection of Bovine Coronavirus by Conventional Reverse Transcription Polymerase Chain Reaction

Cited by 5 publications

References 13 publications

Specific Detection of Bovine Coronavirus N Protein with TaqMan Probe qRT-PCR

Specific Detection of Bovine Coronavirus N Protein with TaqMan Probe qRT-PCR

Advances in Laboratory Diagnosis of Coronavirus Infections in Cattle

Advances in Laboratory Diagnosis of Coronavirus Infections in Cattle

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