title: Rapid and sensitive detection of type II porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip.
abstract: Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was combined with a vertical flow (VF) nucleic acid detection strip to develop a universal assay for the detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). The loop primers were labeled separately with biotin and fluorescein isothiocyanate (FITC) in this assay. Using optimized parameters, the whole reaction could be completed in <50 min in a completely enclosed environment. The detection limit of this assay was found to be 1 pg RNA, 30 tissue culture infective dose 50 (TCID50) virus, or 230 copies of recombinant plasmid DNA, which is relatively higher than that of RT-LAMP analyzed by agarose gel, RT-LAMP visualized by calcein, and the conventional RT-polymerase chain reaction (PCR). No false-positive results were obtained in the specificity assay. The efficiency of the RT-LAMP method was tested by analyzing 43 clinical samples, and the results were compared with those obtained by RT-PCR analysis, with the respective positive rates of 32.56% and 27.91%. This result confirmed that the method described is a rapid, accurate, and sensitive method for universal type II PRRSV detection. Also, this method can be used for the rapid detection of type II PRRSV during the early phase of an outbreak, especially for rapid veterinary diagnosis on the spot and in rural areas.
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
authors.forename: Hongchao
authors.initials: H
authors.lastname: Gou
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
authors.forename: Jieru
authors.initials: J
authors.lastname: Deng
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Pei
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
authors.forename: Jiaying
authors.initials: J
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Liu
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
authors.forename: Mingqiu
authors.initials: M
authors.lastname: Zhao
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. Electronic address: jdchen@scau.edu.cn.
authors.forename: Jinding
authors.initials: J
authors.lastname: Chen
mesh_terms: D000818:Animals; D002845:Chromatography / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005189:False Positive Reactions; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000302:isolation & purification*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D013552:Swine; D013997:Time Factors; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: Loop-mediated isothermal amplification; Porcine reproductive and respiratory syndrome virus; Vertical flow visualization strip