sybr-green-i and Classical-Swine-Fever

Topics: Animals; Benzothiazoles; Circoviridae Infections; Circovirus; Classical Swine Fever; Classical Swine Fever Virus; Diamines; Fluorescence; Nucleic Acid Denaturation; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Swine

A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the rapid and specific detection of HCLV vaccine strain against classical swine fever. Four primers were designed for amplification of NS5B gene region with Bst DNA polymerase at a constant temperature of 65°C. The products showed ladder-like pattern on 2% agarose gel, and can be visualised after addition of SYBR Green I dye. The detection limit of the assay was 5 copies of the HCLV genome per reaction. No cross-reaction with other porcine viruses including different wild-type CSFV strains and the bovine viral diarrhoea virus was observed. The agreement between the LAMP and TaqMan real-time RT-PCR assays was 94.4% for the detection of 72 batches of HCLV vaccine. The assay provides a rapid tool for the control of vaccine quality and can be an accompanying assay of the LAMP for wild-type CSFV described previously for differential diagnosis.

Topics: Animals; Benzothiazoles; China; Classical Swine Fever; Classical Swine Fever Virus; Diagnosis, Differential; Diamines; DNA Primers; Electrophoresis, Agar Gel; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Organic Chemicals; Quinolines; RNA, Viral; Sensitivity and Specificity; Sequence Analysis, DNA; Staining and Labeling; Swine; Temperature; Viral Vaccines; Virology

Classical swine fever is a highly contagious viral disease that causes significant economic losses in pig production on a global scale. The rapid dissemination of the virus and the variability of the clinical signs merit the development of swift and accurate classical swine fever virus (CSFV) detection methods, which can assist in disease control. The development and evaluation of a novel quantitative real-time RT-PCR assay for CSFV detection, based on SYBR Green coupled to melting curve analysis, is described. The analytical and diagnostic performances of the method using two real-time PCR instruments were compared. The assay was specific and detected the major genotypes of CSFV. The limit of detection in cell culture medium and serum was 0.1 TCID50/reaction, while in tissue homogenate for both platforms, it was 1 TCID50/reaction. The limit of detection was 1, 10 and 10² gene copies/μL when nuclease-free water, serum and tissue homogenate, respectively, were used as sample matrices for both instruments. The analysis of 108 tissue homogenate and serum samples from animals infected with CSFV naturally and experimentally and non-infected animals showed that the assay provided a highly sensitive and specific method for classical swine fever.

Topics: Animals; Benzothiazoles; Classical Swine Fever; Classical Swine Fever Virus; Clinical Laboratory Techniques; Diamines; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity; Staining and Labeling; Swine; Virology

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method applied and adapted to the detection of a number of pathogens. A LAMP assay was developed for the specific detection of wild-type classical swine fever virus (CSFV). Based on an alignment of genomic sequences of pestiviruses available in GenBank, four primers were selected targeting six positions in the NS5B gene region of the viral genome. The assay was performed in a simple water bath at a constant temperature of 62 degrees C, and after adding SYBR Green I, the results were visualised by the naked eye. This assay allowed easy applicability in a laboratory that is equipped simply, or even on site, close to the outbreaks and under field conditions. For confirmation, the results could also be visualised under UV light or by separation of the amplified products on 2% agarose gels. The detection limit of the assay was 2.5 medium tissue culture infectious dose (TCID(50)). The assay was validated with 116 clinical samples from vaccinated swineherds and 53 blood samples from experimental infections, and the results were comparable to a real-time RT-PCR assay. In summary, the LAMP assay provides a simple, rapid, and sensitive tool for the detection of wild-type CSFV under field conditions.

Topics: Animals; Benzothiazoles; Classical Swine Fever; Classical Swine Fever Virus; Computational Biology; Diamines; DNA Primers; Nucleic Acid Amplification Techniques; Organic Chemicals; Quinolines; Sensitivity and Specificity; Staining and Labeling; Swine; Temperature; Viral Nonstructural Proteins; Virology