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

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.

Microarray profiling reveals microRNAs involving soybean resistance to Phytophthora sojae.

MicroRNAs (miRNAs), a group of small noncoding RNAs, may serve as a class of post-transcriptional regulators in plant immune systems. Nevertheless, little is known about their roles in plant immune response to the oomycete pathogens. To identify miRNAs involved in the response of soybean to Phytophthora sojae, we examined expressional patterns of miRNAs upon infection by P. sojae by microarray analysis in three soybean cultivars: Williams (susceptible), Conrad (quantitative resistance), and Williams 82 (qualitative resistance). Expression of a number of miRNAs was significantly altered upon infection and (or) in the different genotypes. qRT-PCR data with some miRNAs further confirmed the microarray results. Comparative analysis of the selected miRNAs and their targeted gene expression datasets uncovered many reciprocally expressed miRNA-target pairs, which could proposed a feedback circuit between miRNA(s) and protein-coding genes. These results may serve as a basis for further in-depth studies of miRNAs involved in soybean resistance to P. sojae.