甘蔗赤条病菌巢式PCR检测
Nested-PCR detection of Acidovorax avenae subsp. avenae, the pathogen of red stripe on sugarcane

甘蔗赤条病是由燕麦食酸菌燕麦亚种（Acidovorax avenae subsp. avenae，Aaa）引起的一种世界性甘蔗细菌病害。为建立Aaa快速、灵敏的检测技术，根据该病菌16S~23S核糖体基因及其转录间隔区ITS分别设计2对特异性引物，建立Aaa巢式PCR检测方法。结果表明，建立的巢式PCR方法对Aaa标准菌株、水稻食酸菌A. oryzae具有特异性，可扩增出454 bp目的条带，对近缘种德氏食酸菌A. delafieldii及其它科属的红色雷夫松氏菌Leifsonia rubra和甘蔗宿根矮化病菌L. xyli subsp. xyli未扩增出任何条带。以感染Aaa的甘蔗叶片总DNA、含ITS靶标片段的质粒DNA标准品及Aaa标准菌液为模板，巢式PCR灵敏度最低检测限分别为10 fg/μL、10拷贝/μL和36 CFU/mL，是常规PCR灵敏度的1 000倍。应用巢式PCR和常规PCR对14份有赤条病症状的田间甘蔗叶片样品进行平行检测，阳性检出率分别为100.0%和28.6%，表明巢式PCR比常规PCR检测方法具有更高的灵敏度。本研究建立的巢式PCR方法适合于田间甘蔗赤条病害的分子检测与鉴定。
Red stripe of sugarcane is a worldwide bacterial disease, caused by Acidovorax avenae subsp. avenae (Aaa). To develop a rapid, sensitive nested-PCR assay for Aaa detection in sugarcane, the outer and inner primers were designed based on the nucleotide sequence of 16S-23S ribosomal gene and its internal transcribed spacer (ITS), respectively. Analysis of the primer specificity revealed that the 454 bp targeted band was amplified from Aaa and A. oryzae, but not from the more closely related species A. delafieldii and other bacteria species, namely, Leifsonia rubra and L. xyli subsp. xyli. The nested-PCR assay could detect as little as 10 fg/μL total DNA of sugarcane leaf infected with Aaa, or ten copies/μL DNA of pMD19-Aaa38 plasmids inserted an Aaa 16S-23S ITS fragment, or 36 CFU/mL of Aaa bacterial cells. The sensitivity of nested-PCR assay was 1 000-fold higher than conventional direct-PCR for the three PCR templates tested. Furthermore, fourteen samples of sugarcane leaves with red stripe symptom from the fields were used for the causal pathogen Aaa detection by nested-and direct-PCR, and the detection levels in two methods were 100.0% and 28.6%, respectively. The result indicated that the assay of nested-PCR is an accurate, sensitive, efficient and an alternative tool for the molecular detection and identification of sugarcane red stripe disease.