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      Improved detection of bovine coronavirus N gene in faeces of calves infected naturally by a semi-nested PCR assay and an internal control

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          Abstract

          Bovine coronavirus (BCoV), a positive sense single-stranded RNA virus, is an important causative agent of neonatal diarrhoea in calves from beef and dairy cattle worldwide. The routine detection and diagnosis of BCoV have been mainly dependent on assays with low sensitivity. The aim of the present study was to develop and evaluate a semi-nested PCR (SN-PCR) to amplify a 251 bp fragment of BCoV N gene from fresh ( n = 25) and frozen ( n = 25) diarrhoeic faecal samples of naturally infected calves. To improve detection of BCoV in faecal samples by the SN-PCR an internal control was developed, and the results were compared with a conventional RT-PCR assay. The rates of positive samples by SN-PCR and RT-PCR were 24% (12/50) and 8% (4/50), respectively ( K = 0.43). Only fresh samples were positive in RT-PCR while the SN-PCR detected BCoV in both fresh and frozen faecal samples. The sensitivity of SN-PCR was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) that was detected until 10 −7 dilution. The specificity of the amplicons was assessed by restriction fragment length polymorphism and sequence analysis. The inclusion of an internal control provides a way to detect assay inhibition in faecal samples and failure of nucleic acid extraction that allow reduction of the number of false-negative results.

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          Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model.

          A model was developed to study inhibitors present in feces which prevent the use of PCR for the detection of Helicobacter pylori. A DNA fragment amplified with the same primers as H. pylori was used to spike samples before extraction by a modified QIAamp tissue method. Inhibitors, separated on an Ultrogel AcA44 column, were characterized. Inhibitors in feces are complex polysaccharides possibly originating from vegetable material in the diet.
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            Bovine coronavirus

            M.A Clark (1993)
            Summary This review aims to summarize current data describing the characteristics of bovine coronavirus (BCV) and the three clinical syndromes with which this virus is associated. The first half of this paper consists of a general description of the virus, commencing with a brief outline of the methods used for in vitro growth. The structure of the virus is then described in more detail, with particular reference to the structure and functions of the four major viral proteins. This is followed by an outline of the unique replication strategy adopted by coronaviruses. The second half of this review discusses the clinical significance of the virus, beginning with a detailed account of BCV-induced neonatal calf diarrhoea, the clinical syndrome with which this virus is most commonly associated. The clinical and epidemiological importance of BCV respiratory tract infection is then discussed, and finally the evidence supporting the aetiological role of BCV in outbreaks of winter dysentery in adult cattle is examined.
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              Practical considerations in design of internal amplification controls for diagnostic PCR assays.

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                Author and article information

                Contributors
                Journal
                J Virol Methods
                J. Virol. Methods
                Journal of Virological Methods
                Elsevier B.V.
                0166-0934
                1879-0984
                22 September 2005
                February 2006
                22 September 2005
                : 131
                : 2
                : 148-154
                Affiliations
                Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina (UEL). Campus Universitário, PO Box 6001, 86051-990, Londrina, Paraná, Brazil
                Author notes
                [* ]Corresponding author. Tel.: +55 43 3371 4485; fax: +55 43 3371 4714. alfieri@ 123456uel.br
                Article
                S0166-0934(05)00265-X
                10.1016/j.jviromet.2005.08.005
                7112777
                16182383
                d9270517-8428-4e07-8b90-13bf1bbff33f
                Copyright © 2005 Elsevier B.V. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 22 April 2005
                : 12 August 2005
                : 18 August 2005
                Categories
                Article

                Microbiology & Virology
                calves,diarrhoea,bovine coronavirus,sn-pcr
                Microbiology & Virology
                calves, diarrhoea, bovine coronavirus, sn-pcr

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