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Open Access Methodology

One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification

Qi-Wei Wei1, Cui Yu2, Shu-Ya Zhang2, Cui-Yun Yang2, Karwitha Miriam1, Wen-Na Zhang1, Dao-Long Dou1 and Xiao-Rong Tao1*

Author Affiliations

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

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Virology Journal 2012, 9:187  doi:10.1186/1743-422X-9-187

Published: 7 September 2012

Abstract

Background

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.

Methods

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.

Results

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.

Conclusion

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.

Keywords:
Bean pod mottle virus (BPMV); RT-LAMP; Rapid detection; Soybean seeds