Abstract False-negative result is a major factor affecting the results of nucleic acid testing. The internal amplification control (IAC) plays an important role in monitoring false negative results during PCR. In order to solve the false-negative result, the application of the IAC in reverse transcription- recombinase polymerase amplification (RT-RPA) was established. The IAC was constructed by the composite primer method and applied to the detection of three soybean viruses, Soybean mosaic virus (SMV), Bean pod mottle virus (BPMV) and Southern bean mosaic virus (SBMV), during RT-RPA. The results showed that the methods were able to achieve specific amplification of the target gene while indicating false negative results. Sensitivity experiments showed that the detection limit of this assay for SMV and BPMV was 0.05 ng, when the content of the IAC was 1.83×104 copies in 50 μL reaction system. And when the content of the IAC was 1.83×103 copies in 50 μL reaction system, the detection limit of this assay for SBMV was 0.05 ng. The methods were used to test 10 soybean (Glycine max) samples and the results were consistent with those of the corresponding reverse transcription-PCR (RT-PCR), indicating its good applicability. In conclusion, the IAC-RT-PRA methods for SMV, BPMV and SBMV were established in this study could indicate false negatives and improve credibility of test results.
YUAN Jun-Jie,LONG Yang,WEI Ting-Yu, et al. RT-RPA Technology Included with IAC for Detection of Three Soybean (Glycine max) Viruses[J]. 农业生物技术学报, 2020, 28(12): 2261-2269.
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