Abstract Dendrobium officinale is a valuable and rarely Chinese herb, which is easy to suffer from various diseases during its growth. Thus early diagnosis of those diseases is crucial to increase the yield of D. officinale. In this study, we compared the internal transcribed spacer regions of the ribosomal DNA sequences of four widely disseminated pathogens in D. officinale (Sclerotium delphinii, Colletotrichum gloeosporioides, Alternaria alternata, and Cladosporium cladosporioides), designed specific primers for A. alternata and C. cladosporioides, and established a four-seat nested multiplex PCR assay. The sensitivity for S. delphinii, C. cladosporioides, A. alternata was found to be 10 fg/μL, and 10 pg/μL for C. gloeosporioides. In the actual application test, our established nested multiplex PCR can detect pathogens more quickly and accurately than conventional culture method. This method combines the advantages of nested and multiplex techniques, and it considerably improves the sensitivity and specificity of the detection results. Therefore, we hope that this method can provide a tool for diagnosing D. officinale diseases.

中文翻译：

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巢式多重PCR法同时检测铁皮石斛四种病原菌

摘要 铁皮石斛是一种名贵珍稀的中草药，在其生长过程中易患多种病害。因此，这些疾病的早期诊断对于提高铁皮茶的产量至关重要。在这项研究中，我们比较了铁皮石斛中四种广泛传播的病原体（飞燕核、胶孢炭疽菌、交链孢菌和枝孢枝孢菌）的核糖体 DNA 序列的内部转录间隔区，并设计了链孢霉和 C. cladosporioides，并建立了一个四座巢式多重 PCR 检测。发现 S. delphinii、C. cladosporioides、A. alternata 的灵敏度为 10 fg/μL，而 C. gloeosporioides 的灵敏度为 10 pg/μL。在实际应用测试中，我们建立的巢式多重 PCR 可以比传统的培养方法更快、更准确地检测病原体。该方法结合了嵌套和多重技术的优点，大大提高了检测结果的灵敏度和特异性。因此，我们希望该方法能够为铁皮石斛疾病的诊断提供一个工具。