Efficient quantification of Globodera pallida and G. rostochiensis (Tylenchida: Heteroderidae) in large amounts of soil using probe-based real-time PCR

IF 1.3 4区 农林科学 Q2 ENTOMOLOGY Applied Entomology and Zoology Pub Date : 2024-02-24 DOI:10.1007/s13355-024-00863-y
Itaru Sakata, Kenji Itou, Atsuhiko Kushida
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Abstract

Real-time quantitative polymerase chain reaction (qPCR) is used to estimate the population densities of the potato cyst nematodes Globodera pallida Stone and Globodera rostochiensis (Wollenweber) Skarbilovich (Tylenchida: Heteroderidae). Since it is difficult to extract nematode DNA from large amounts of soil (≥ 100 g, enough for quantification of cyst nematodes), cyst isolation is required before DNA extraction. However, when isolating cysts from the soil, various impurities are simultaneously isolated, and separating the cysts from these impurities is laborious. Although previous studies have reported methods for extracting DNA from mixtures of cysts and impurities, it is unclear whether such DNA can be used to estimate nematode densities using qPCR. To examine the effects of impurities on the accuracy of qPCR quantification, we extracted DNA from nematode eggs (G. pallida and G. rostochiensis) mixed with impurities and performed qPCR. The results suggested that the differences in the fields affected the quantification accuracy. Therefore, field-specific standard curves should be set, which are impractical for routine diagnosis. To propose a more practical method, we determined a fixed standard curve for each species and estimated the population densities in field soil samples by qPCR using the standard curves. The estimated population densities significantly correlated with those determined using conventional microscopic inspections. This study revealed that the population densities of G. pallida and G. rostochiensis can be estimated from large amounts of soil, probably only approximately, but efficiently, by qPCR using DNA extracted from mixtures of cysts and impurities.

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利用基于探针的实时 PCR 高效定量检测大量土壤中的 Globodera pallida 和 G. rostochiensis(天牛科:异足目)含量
实时定量聚合酶链式反应(qPCR)用于估算马铃薯胞囊线虫 Globodera pallida Stone 和 Globodera rostochiensis (Wollenweber) Skarbilovich(Tylenchida: Heteroderidae)的种群密度。由于很难从大量土壤中提取线虫 DNA(≥ 100 克,足以对包囊线虫进行定量),因此在提取 DNA 之前需要先分离包囊。然而,从土壤中分离孢囊时,会同时分离出各种杂质,将孢囊从这些杂质中分离出来非常费力。虽然之前的研究已经报道了从孢囊和杂质的混合物中提取 DNA 的方法,但目前还不清楚这些 DNA 是否可用于使用 qPCR 估算线虫密度。为了研究杂质对 qPCR 定量准确性的影响,我们从混有杂质的线虫卵(G. pallida 和 G. rostochiensis)中提取 DNA 并进行 qPCR。结果表明,田间差异影响了定量的准确性。因此,应设定田间特异性标准曲线,但这在常规诊断中并不实用。为了提出一种更实用的方法,我们为每个物种确定了一条固定的标准曲线,并利用标准曲线通过 qPCR 估算了田间土壤样本中的种群密度。估算出的种群密度与传统显微镜检查确定的种群密度有明显的相关性。这项研究表明,通过使用从包囊和杂质混合物中提取的 DNA 进行 qPCR,可以从大量土壤中估算出 G. pallida 和 G. rostochiensis 的种群密度,虽然只能估算出大概的数值,但却非常有效。
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来源期刊
CiteScore
2.70
自引率
7.70%
发文量
37
审稿时长
6-12 weeks
期刊介绍: Applied Entomology and Zoology publishes articles concerned with applied entomology, applied zoology, agricultural chemicals and pest control in English. Contributions of a basic and fundamental nature may be accepted at the discretion of the Editor. Manuscripts of original research papers, technical notes and reviews are accepted for consideration. No manuscript that has been published elsewhere will be accepted for publication.
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