title: Establishment of a sensitive and specific hyper-branched rolling circle amplification assay and test strip for TSV.
abstract: A specific padlock probe (PLP) and detection probe were designed to target the capsid protein gene of Taura syndrome virus (TSV), and a hyper-branched rolling circle amplification (HRCA) assay and a corresponding strip-based test were produced. The reaction time and temperature were optimized for both. The PLPs were linked with the target sequence via T4 DNA ligase under conditions of 37°C for 30 min, followed by reaction with Bst DNA polymerase Large Fragment at 61°C for 30 min, and then the test strip was made using the detection probe. Both the assay and the strip had similarly high accuracy and specificity, and their sensitivity was close to 10 copies, which is 100 times higher than that of conventional RT-PCR. We tested 89 batches of shrimps for TSV to assess the HRCA assay and test strip; the results indicated that the TSV HRCA assay and the test strip are rapid diagnostic tools for detection in the field, and have potential for early diagnosis of TSV.
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China; Technical Center of Shandong Inspection and Quarantine Bureau, Qingdao 266002, China.
authors.forename: Yuran
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Yantai Inspection and Quarantine Bureau, Yantai 26400, China.
authors.forename: Weili
authors.initials: W
authors.lastname: Yin
authors.affiliation: Qingdao Agriculture University, College of Food Science and Engineering, Qingdao 266109, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Technical Center of Shandong Inspection and Quarantine Bureau, Qingdao 266002, China.
authors.forename: Gongpu
authors.initials: G
authors.lastname: Wang
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China. Electronic address: bfli@ouc.edu.cn.
authors.forename: Bafang
authors.initials: B
authors.lastname: Li
mesh_terms: D000818:Animals; D036022:Capsid Proteins / Q000235:genetics*; D056629:Dicistroviridae / Q000235:genetics / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes; D033561:Penaeidae / Q000821:virology; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D036022:Capsid Proteins; D015345:Oligonucleotide Probes
keywords: HRCA; HRCA test strip; PLP; RT-PCR; Shrimp; TSV