期刊
VIROLOGY JOURNAL
卷 9, 期 -, 页码 -出版社
BMC
DOI: 10.1186/1743-422X-9-187
关键词
Bean pod mottle virus (BPMV); RT-LAMP; Rapid detection; Soybean seeds
类别
资金
- National High Technology Research and Development Program of China (863 Program) [2012AA101501]
- National Natural Science Foundation of China [30900936, 31171813]
- Fundamental Research Funds for the Central Universities [KYZ201001]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
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 degrees 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.
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