One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification.
One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification.
复制标题
逆转录环介导等温扩增一步法检测大豆种子中豆荚斑驳病毒
DOI：
10.1186/1743-422x-9-187
复制
发表时间：
2012-09-07
期刊：
影响因子：
4.8
通讯作者：
Tao XR
中科院分区：
文献类型：
--
作者：
Wei QW;Yu C;Zhang SY;Yang CY;Miriam K;Zhang WN;Dou DL;Tao XR
背景
豆荚斑驳病毒（Bean pod mottle virus，BPMV）是一种传播范围广、危害性大的病毒，每年在许多国家造成巨大的经济损失。因此，迫切需要一种灵敏、可靠、特异的快速监测方法，以防止BPMV的进一步传播。
方法
在BPMV CP基因保守区设计了一对简并逆转录环介导等温扩增（RT-LAMP）引物。优化了RT-LAMP的反应条件，并以快速提取的大豆种子RNA粗品为材料，评价了该方法检测BPMV的可行性、特异性和敏感性。
结果
优化的RT-LAMP反应条件为6 mM MgCl 2、0.8M甜菜碱和62.5-65°C，可成功地从BPMV感染的大豆种子中扩增出梯状条带。扩增产物对BPMV具有很强的特异性，与其它大豆病毒无交叉反应。荧光染料的内含物使其易于在管内通过肉眼检测。结果表明，在本试验条件下，RT-LAMP方法的灵敏度高于常规RT-PCR方法，常规RT-PCR方法不能用于大豆种子RNA粗提物的检测。
结论
通过快速RNA提取和RT-LAMP相结合，建立了一种高效实用的检测大豆种子中BPMV的方法。这种RT-LAMP方法具有快速监测BPMV的巨大潜力，并将有助于防止这种毁灭性病毒的进一步传播。
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.