[Development and Application of Reverse Transcription Loop-mediated Isothermal Amplification Method for Detection of Porcine Rotavirus of Swine].

Abstract

We wished to establish a method for rapid and sensitive detection of reverse transcription loop-mediated isothermal amplification(RT-LAMP)for the rapid and sensitive detection of porcine rotavirus (PoRV). According to the published PoRV VP7 sequences in GenBank,6specific primers were designed. According to the concentrations of foward and reverse primers, Bst DNA polymerase, Mg(2+), and dNTP, reaction conditions were optimized. Results revealed the concentration ratio of foward and reverse primers to be 200 nmol/L:2, 400 nmol (1:12), Bst DNA polymerase concentration to be 0.64U/μL,Mg2+concentration to be 2.5mmol/L, and dNTP concentration to be 1.0mmol/L in 1hat 60℃.The amplification effect achieved a "ladder" effect, with amplified bands being shown only for PoRV. RT-LAMP was specific and did not elicit a cross reaction with porcine epidemic diarrhea virus, transmissible gastroenteritis virus of pigs, or classical swine fever virus. The sensitivity of RT-LAMP was 1.0×10(2) copies/μL. After the reaction, inspection by the naked eye revealed positive amplification products to appears as cloudy-white precipitates, and addition of SYBR Green I showed a color change. These data demonstrate that RT-LAMP is suitable for the rapid and sensitive detection of PoRV.