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

Sep 22, 2014Journal of virological methods

Fast and Accurate Detection of Type II Swine Reproductive and Respiratory Virus Using a Simple Amplification and Visual Test

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Abstract

The detection limit of the developed assay for type II porcine reproductive and respiratory syndrome virus (PRRSV) is 1 pg RNA.

The assay utilizes reverse transcription-loop-mediated isothermal amplification (RT-LAMP) combined with a vertical flow nucleic acid detection strip.
The entire reaction can be completed in less than 50 minutes within a closed environment.
No false-positive results were observed during the specificity testing.
In a comparison of clinical samples, the assay showed a positive rate of 32.56%, while RT-PCR showed a rate of 27.91%.
The method is indicated as rapid, accurate, and sensitive for detecting type II PRRSV, particularly useful during early outbreak phases.

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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.

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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

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