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Polymerase chain reaction combined with fluorescent lateral flow immunoassay based on magnetic purification for rapid detection of canine parvovirus 2.

ABSTRACT: BACKGROUND:Canine parvovirus 2 (CPV-2) is one of the most common etiological agents that cause severe gastroenteritis in puppies. Early accurate diagnosis is important for infected dogs. In recent years, magnetic separation has become an efficient and useful tool for bioassays. In this study, polymerase chain reaction (PCR) combined with fluorescent lateral flow immunoassay (LFIA) based on magnetic purification assay was developed for the quantitative detection of CPV-2. RESULTS:The optimum working reaction volume and reaction time for LFIA was 100??L and 2?min, respectively. The PCR-LFIA assay only detected CPV-2, and did not show cross-detection of non-CPV strains. Experiments showed analytical sensitivity of 3?×?101 copies/?L and demonstrated the PCR-LFIA has a diagnostic agreement of 100% with conventional PCR on detection of clinical samples (22.6% positive, 14/62). Cutoff value is 146. The results were further verified by sequencing and BLAST software. The entire process from PCR step only takes ~?80?min. CONCLUSIONS:This approach provides an attractive platform for rapid and quantitative detection of CPV-2, indicating great promise as a convenient molecular detection tool to facilitate disease outbreak investigations and response timely.

SUBMITTER: Zhuang L

PROVIDER: S-EPMC6337814 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Polymerase chain reaction combined with fluorescent lateral flow immunoassay based on magnetic purification for rapid detection of canine parvovirus 2.

Zhuang Linlin L Ji Yongxin Y Tian Peilong P Wang Kaixuan K Kou Chengkun C Gu Ning N Zhang Yu Y

BMC veterinary research 20190117 1

<h4>Background</h4>Canine parvovirus 2 (CPV-2) is one of the most common etiological agents that cause severe gastroenteritis in puppies. Early accurate diagnosis is important for infected dogs. In recent years, magnetic separation has become an efficient and useful tool for bioassays. In this study, polymerase chain reaction (PCR) combined with fluorescent lateral flow immunoassay (LFIA) based on magnetic purification assay was developed for the quantitative detection of CPV-2.<h4>Results</h4>T ...[more]

PMID: 30654823

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Project description:A fluorescent immunoassay based on europium nanoparticles (EuNPs-FIA) was developed for the simultaneous detection of antibiotic residues, solving the problems of single target detection and low sensitivity of traditional immunoassay methods. In the EuNPs-FIA, EuNPs were used as indictive probes by binding to anti-tetracyclines monoclonal antibodies (anti-TCs mAb), anti-sulphonamides monoclonal antibodies (anti-SAs mAb) and anti-fluoroquinolones monoclonal antibodies (anti-FQs mAb), respectively. Different artificial antigens were assigned to different regions of the nitrocellulose membrane as capture reagents. The EuNPs-FIA allowed for the simultaneous detection of three classes of antibiotics (tetracyclines, fluoroquinolones and sulphonamides) within 15 min. It enabled both the qualitative determination with the naked eye under UV light and the quantitative detection of target antibiotics by scanning the fluorescence intensity of the detection probes on the corresponding detection lines. For qualitative analysis, the cut-off values for tetracyclines (TCs), fluoroquinolones (FQs) and sulphonamides (SAs) were 3.2 ng/ml, 2.4 ng/ml and 4.0 ng/ml, respectively, which were much lower than the maximum residue limit in food. For quantitative analysis, these ranged from 0.06 to 6.85 ng/ml for TCs, 0.03-5.14 ng/ml for FQs, and 0.04-4.40 ng/ml for SAs. The linear correlation coefficients were higher than 0.97. The mean spiked recoveries ranged from 92.1 to 106.2% with relative standard deviations less than 8.75%. Among them, the three monoclonal antibodies could recognize four types of TCs, seven types of FQs and 13 types of SAs, respectively, and the detection range could cover 24 antibiotic residues with different structural formulations. The results of the detection of antibiotic residues in real samples using this method were highly correlated with those of high performance liquid chromatography (R 2 > 0.98). The accuracy and precision of the EuNPs-FIA also met the requirements for quantitative analysis. These results suggested that this multiplex immunoassay method was a promising method for rapid screening of three families of antibiotic residues.

Dataset Information

Polymerase chain reaction combined with fluorescent lateral flow immunoassay based on magnetic purification for rapid detection of canine parvovirus 2.

Publications

Polymerase chain reaction combined with fluorescent lateral flow immunoassay based on magnetic purification for rapid detection of canine parvovirus 2.

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