Rapid and Sensitive Detection of Bovine Coronavirus and Group A Bovine Rotavirus from Fecal Samples by Using One-Step Duplex RT-PCR Assay

Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

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Detection of bovine corona virus in some governorates of Iraq

Al-Qadisiyah Journal of Veterinary Medicine Sciences ◽

10.29079/vol12iss1art225 ◽

2013 ◽

Vol 12 (1) ◽

pp. 24

Author(s):

Kh. A. Mansour

Keyword(s):

Polymer Chain ◽

Reverse Transcription ◽

Rt Pcr ◽

Chain Reaction ◽

Pcr Assay ◽

Bovine Coronavirus ◽

Fecal Samples ◽

Clinically Normal ◽

Corona Virus ◽

Diarrheic Calves

In the present study, we used Reverse transcription polymer chain reaction (RT-PCR) assay for detecting of bovine coronavirus, We evaluated the presence of bovine coronavirus ( BCoV) in diarrheic fecal samples in age (1 – 30) day. (152) fecal samples from diarrheic calves were collected from four Iraqi governorates (Al-Qadisyiah, Babylon, Wassit, and Najaf) .10 (6.57%) out of 152 were positive to bovine coronavirus. This study is the first detection of bovine corona antigen in Iraq.T he results suggest that RT-PCR is more sensitive than another method to detect BCoV, especially in subclinical cases and Because these animals shed a low amount of virus in feces and more animal show clinically normal we need to apply sensitive techniques, such as RT-PCR.

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Development of a one-step RT-PCR assay for detection of pancoronaviruses (α-, β-, γ-, and δ-coronaviruses) using newly designed degenerate primers for porcine and avian `fecal samples

Journal of Virological Methods ◽

10.1016/j.jviromet.2018.02.021 ◽

2018 ◽

Vol 256 ◽

pp. 116-122 ◽

Cited By ~ 14

Author(s):

Hui Hu ◽

Kwonil Jung ◽

Qiuhong Wang ◽

Linda J. Saif ◽

Anastasia N. Vlasova

Keyword(s):

Rt Pcr ◽

Pcr Assay ◽

Degenerate Primers ◽

Fecal Samples ◽

One Step

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PCR Detection of Group A Bovine Rotaviruses in Feces

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879300500403 ◽

1993 ◽

Vol 5 (4) ◽

pp. 516-521 ◽

Cited By ~ 11

Author(s):

Jarasvech Chinsangaram ◽

Geoffrey Y. Akita ◽

Anthony E. Castro ◽

Bennie I. Osburn

Keyword(s):

Ethidium Bromide ◽

Clinical Samples ◽

Pcr Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Pcr Assay ◽

Fecal Samples ◽

Bovine Rotavirus ◽

Viral Particles ◽

Pcr Inhibitor ◽

Group A

A polymerase chain reaction (PCR) protocol has been developed for identification of bovine group A rotavirus infection in feces. Primers (20mers) complementary to 3′ ends of double-stranded RNA genome segment 6 of bovine rotavirus NCDV strain were synthesized and used in PCR. Bovine rotavirus RNA from infected cell culture was employed to optimize the PCR protocol. Rotavirus-negative fecal samples were spiked with known quantities of bovine rotavirus, and the sensitivity of the PCR assay was determined. Fecal samples were extracted with phenol and treated to eliminate unidentified PCR inhibitor(s) in feces, and PCR was performed. PCR products were either visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the assay was 6 × 104 viral particles/ml of feces with ethidium bromide-stained agarose gel visualization or 6 × 102 viral particles/ml of feces with chemiluminescent hybridization. The PCR assay was applied to 18 fecal specimens from clinical cases. All 16 clinical samples that were positive for rotavirus by enzyme-linked immunosorbent assay (ELISA) or by ELISA and electron microscopy (EM) were positive by PCR. The 2 samples that were rotavirus negative by ELISA or by ELISA and EM were also negative on PCR analysis.

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Sensitive Detection and Quantification of SARS-CoV-2 by Multiplex Droplet Digital RT-PCR

10.21203/rs.3.rs-81304/v1 ◽

2020 ◽

Author(s):

Remco de Kock ◽

Mieke Baselmans ◽

Volkher Scharnhorst ◽

Birgit Deiman

Keyword(s):

Simultaneous Detection ◽

Housekeeping Gene ◽

Multiplex Assay ◽

Viral Rna ◽

Sensitive Detection ◽

Rt Pcr ◽

Pcr Assay ◽

Assay Sensitivity ◽

One Step ◽

Detection And Quantification

Abstract Purpose. We aimed to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive detection of SARS-CoV-2 RNA with respect to human derived RNA and could be used for triage and monitoring of Covid-19 patients. Methods. A one step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19 negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. Results. Assay sensitivity of the RT-PCR assay drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared to water, while the sensitivity of the RT-ddPCR was not affected by the total NA background. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. Conclusion. The present study describes a robust and sensitive one-step RT-ddPCR multiplex assay for reliable detection and quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.

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Bovine coronavirus detection in a collection of diarrheic stool samples positive for group a bovine rotavirus

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132009000700006 ◽

2009 ◽

Vol 52 (spe) ◽

pp. 45-49 ◽

Cited By ~ 8

Author(s):

Aline Fernandes Barry ◽

Alice Fernandes Alfieri ◽

Danilo Tancler Stipp ◽

Amauri Alcindo Alfieri

Keyword(s):

Economic Losses ◽

Bovine Coronavirus ◽

Fecal Samples ◽

Nucleoprotein Gene ◽

Bovine Rotavirus ◽

Unusual Event ◽

Group A Rotaviruses ◽

Group A ◽

Stool Samples ◽

Viral Etiologies

Neonatal diarrhea is an important cause of economic losses for cattle farmers. The main viral etiologies of enteric diseases are group A rotaviruses (GARV) and the bovine coronavirus (BCoV). Although both viruses infect calves of the same age, the occurrence of mixed infections is still under studied. The present study describes the co-infection of BCoV and GARV in stool samples. Forty-four diarrheic fecal samples from calves up to 60 days old that had previously tested positive for GARV by SS-PAGE were analyzed using semi-nested PCR for BCoV. A product with 251 bp of the BCoV nucleoprotein gene was amplified in 15.9% (7/44) of the samples, demonstrating that co-infection is not an unusual event. These results reinforce the need for testing for both GARV and BCoV, even in fecal samples that previously tested positive for one virus.

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