One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification
1 Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China
2 Shanghai Entry Exit Inspection and Quarantine Bureau, Shanghai 200135, People’s Republic of China
Virology Journal 2012, 9:187 doi:10.1186/1743-422X-9-187Published: 7 September 2012
Bean pod mottle virus (BPMV) is a wide-spread and destructive virus that causes huge economic losses in many countries every year. A sensitive, reliable and specific method for rapid surveillance is urgently needed to prevent further spread of BPMV.
A degenerate reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer set was designed on the conserved region of BPMV CP gene. The reaction conditions of RT-LAMP were optimized and the feasibility, specificity and sensitivity of this method to detect BPMV were evaluated using the crude RNA rapidly extracted from soybean seeds.
The optimized RT-LAMP parameters including 6 mM MgCl2, 0.8 M betaine and temperature at 62.5-65°C could successfully amplify the ladder-like bands from BPMV infected soybean seeds. The amplification was very specific to BPMV that no cross-reaction was observed with other soybean viruses. Inclusion of a fluorescent dye makes it easily be detected in-tube by naked eye. The sensitivity of RT-LAMP assay is higher than the conventional RT-PCR under the conditions tested, and the conventional RT-PCR couldn’t be used for detection of BPMV using crude RNA extract from soybean seeds.
A highly efficient and practical method was developed for the detection of BPMV in soybean seeds by the combination of rapid RNA extraction and RT-LAMP. This RT-LAMP method has great potential for rapid BPMV surveillance and will assist in preventing further spread of this devastating virus.