Testing new PCR primers and a TaqMan™ probe for detection of Phlyctema vagabunda syn. Neofabraea alba

Q3 Agricultural and Biological Sciences

New Zealand Plant Protection Pub Date : 2019-07-28 DOI:10.30843/NZPP.2019.72.309

M. J. Vergara, I. Pushparajah, J. Mackay, K. Everett

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引用次数: 0

Abstract

Phlyctema vagabunda syn. Neofabraea alba is a fungal pathogen that causes bull’s eye rot (BER) of apples. Polymerase chain reaction (PCR) primers complementary to the inter-transcribed spacer region of ribosomal DNA (ITS) and the β-tubulin gene region, and a TaqMan™ probe assay were developed to detect this pathogen. These assays were compared in quantitative PCR (qPCR) reactions for amplification of DNA extracted from several fungal species and from apple tissue. Although the ITS and the β-tubulin primers amplified all N. alba isolates, both primers also amplified a few other fungal species. The TaqMan™ probe used with published primers for N. alba only amplified N. alba isolates. The TaqMan™ assay resulted in the lowest crossing threshold (Ct) values for DNA extracted from apple fruit, leaves, and spores collected on cellophane from eight apple orchards. The TaqMan™ results were correlated with percentage BER (%BER) in a 400-apple sample harvested from the same orchards. The TaqMan™ probe assay was the most sensitive and specific qPCR protocol tested, and Ct values showed the best correlation with %BER.

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新PCR引物和TaqMan™探针检测斑疹伤寒

白腐霉是一种引起苹果牛眼腐病的真菌病原体。建立了核糖体DNA (ITS)转录间隔区和β-微管蛋白基因区互补的聚合酶链反应(PCR)引物，以及TaqMan™探针法检测该病原体。用定量PCR (qPCR)对几种真菌和苹果组织中提取的DNA进行扩增。虽然ITS和β-微管蛋白引物扩增了所有白僵菌，但这两种引物也扩增了其他几种真菌。TaqMan™探针与已发表的引物一起用于白僵菌，仅扩增白僵菌分离株。TaqMan™检测结果显示，从8个苹果园的苹果果实、叶片和孢子中提取的DNA具有最低的交叉阈值(Ct)。TaqMan™结果与从同一果园收获的400个苹果样品的BER百分比(%BER)相关。TaqMan™探针检测是最敏感和特异的qPCR检测方案，Ct值与%BER的相关性最好。

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来源期刊

New Zealand Plant Protection Agricultural and Biological Sciences-Insect Science

CiteScore

1.10

自引率

0.00%

发文量

期刊介绍： New Zealand Plant Protection is the journal of the New Zealand Plant Protection Society. It publishes original research papers on all aspects of biology, ecology and control of weeds, vertebrate and invertebrate pests, and pathogens and beneficial micro-organisms in agriculture, horticulture, forestry and natural ecosystems of relevance to New Zealand.