甘蔗宿根矮化病菌巢式PCR检测

Nested-PCR detection for Leifsonia xyli xyli,the causal bacterium of sugarcane ratoon stunting disease

为了探索甘蔗宿根矮化病快速检测技术,以细菌16S-23S rDNA内转录间隔区(ITS)通用引物ITS1/ITS2为第1轮引物,以甘蔗宿根矮化病菌特异引物Lxx1/Lxx2为第2轮引物,建立甘蔗宿根矮化病菌巢式PCR检测技术。巢式PCR能扩增出438 bp的目的条带,其核苷酸序列与巴西、澳大利亚及美国甘蔗宿根矮化病菌分离物核苷酸序列同源率为100%或99.8%;其检测灵敏度为10fg甘蔗宿根矮化病菌基因组DNA,较常规PCR提高100倍;样品检测结果也表明巢式PCR检测灵敏度明显优于常规PCR,可用于+1片嫩叶和心叶等微量病菌样品的检测。

In order to develop quick and sensitive detection technique for the causal agent of ratoon stun- ting disease of sugarcane, Leifsonia xyli xyli, this study developed a nested-PCR for rapid and accurate detection of L. xyli xyli using bacterial universal primer pair ITS1/ITS2, based on the intergenic tran- scribed spacer （ITS） region of 16S-23S ribosomal DNA of bacterium, as the first-round primers followed by a L. xyli xyli specific primer pair Lxxl/Lxx2. The nested-PCR could amplify a unique 438 bp specific fragment whose nucleotide sequence sharing 100 % and 99.8 % nucleotide identity with Brazil, Austral- ia and USA isolates of L. xyli xyli, and its detection sensitivity was 10 fg of L. xyli xyli genomic DNA in 20 p.L reaction solution, which increased the detection sensitivity by 100-fold compared to the simple PCR method. Detection results from samples also showed that detection sensitivity of the nested-PCR was sig- nificantly better than that of the simple PCR. The nested-PCR could be used to detect L. xyli xyli from some sugarcane tissue with very few L. xyli xyli, such as ＋ 1 young leaf and the youngest leaf above grow- ing point.