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为准确、快速测定感病柑橘花器和种子中黄龙病菌(Candidatus Liberibacter asiaticus,Las)的含量,比较了SYBR GreenⅠ实时荧光定量PCR(SGI-qPCR)和单管双引物对TaqMan探针qPCR(STDP-qPCR)的检测灵敏度,并用STDP-qPCR法定量检测了感病沙田柚花器和种子中的Las。结果显示,STDP-qPCR检测灵敏度为1×100拷贝/μL,比SGI-qPCR高100倍;花器中的雄蕊、花瓣、雌蕊和花粉等组织,以及种子的种皮和胚乳组织中均可检测到Las,但含量差异较大,其中种皮组织中Las含量最高,达到109842个细胞/μg DNA,花粉中的Las含量最低,为308个细胞/μg DNA,所有种壳中均未检测到Las;基于雄蕊组织的黄龙病分子诊断准确率达93.8%。表明Las在感病柑橘花器和种子中呈不均匀分布,基于雄蕊组织的黄龙病诊断方法可辅助用于该病害的高通量检测。
In order to accurately and rapidly determine the content of Candidatus Liberibacter asiaticus (Lasius) in susceptible citrus flowers and seeds, SYBR Green Ⅰ real-time fluorescence quantitative PCR (SGI-qPCR) and single tube double primer were used to detect TaqMan probe qPCR (STDP- qPCR), and the detection of Las in the flower and seed of susceptible Shaddock (Citrus reticulata) by STDP-qPCR. The results showed that the detection sensitivity of STDP-qPCR was 1 × 100 copies / μL, which was 100 times higher than that of SGI-qPCR. The stamens, petals, pistils and pollens in flower organs and the seed coat and endosperm tissues were all detected Las, but the contents were quite different. The content of Las was the highest in the seed coat tissues, reaching 109842 cells / μg DNA. The content of Las in the pollen was the lowest at 308 cells / μg DNA. No Las was detected in all the seed shells. Based on the stamens tissue molecular diagnosis Huanglongbing accuracy rate of 93.8%. Indicating that Las was unevenly distributed in susceptible citrus flowers and seeds. The diagnostic method based on stamens of the yellow dragon’s disease could be used for the high-throughput detection of this disease.