Rapid detection of porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification.
The fragment of the membrane protein M gene with high conservation and specificity of porcine reproductive and respiratory syndrome virus (PRRSV) was chosen to be the target region, according to which six special primers were designed successfully. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to detect the PRRSV by incubation at 65 degrees for only 45 min with the ladder-like pattern of bands from 182 bp on the agarose gel, and the product specificity was confirmed by RsaI. The result of RT-LAMP could also be visualized directly with the naked eye by adding the intercalating dye Picogreen. The RT-LAMP was identified to detect only the PRRSV in all viruses tested, which demonstrated the high specificity. By using various sample dilutions as templates, the sensitivity of RT-LAMP was found to be 100-fold higher than that of RT-PCR and could be comparable to the fluorescence quantitative RT-PCR. A comparison was obtained by the RT-LAMP and PCR assays using 20 clinical samples. Finally, a rapid, convenient and reliable PRRSV detection system was developed using the RT-LAMP.
Qin
Chen
C
School of Life Science, Shanghai University, Shanghai, People's Republic of China. chenqincc@yahoo.com.cn
Jian
Li
L
Xue-En
Fang
F
Wei
Xiong
X
D000818:Animals; D001769:Blood / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D008168:Lung / Q000821:virology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013552:Swine; D013696:Temperature; D013997:Time Factors; D014763:Viral Matrix Proteins / Q000235:genetics
D017931:DNA Primers; C525999:M protein, porcine reproductive and respiratory syndrome virus; D009930:Organic Chemicals; C099476:PicoGreen; D014763:Viral Matrix Proteins