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用于家鸡皮刺螨(Dermanyssus gallinae)定量PCR分析的内参基因选择

Selection of reference genes for quantitative PCR analysis in poultry red mite (Dermanyssus gallinae).

作者信息

Ariizumi Takuma, Murata Shiro, Fujisawa Sotaro, Isezaki Masayoshi, Maekawa Naoya, Okagawa Tomohiro, Sato Takumi, Oishi Eiji, Taneno Akira, Konnai Satoru, Ohashi Kazuhiko

机构信息

Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Kita-18, Nishi-9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan.

Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Kita-18, Nishi-9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan.

出版信息

J Vet Med Sci. 2021 Apr 9;83(4):558-565. doi: 10.1292/jvms.20-0677. Epub 2021 Feb 11.

Abstract

Poultry red mites (PRMs, Dermanyssus gallinae) are harmful ectoparasites that affect farmed chickens and cause serious economic losses in the poultry industry worldwide. Acaricides are used for PRM control; however, some PRMs have developed acaricide-resistant properties, which have indicated the need for different approaches for PRM control. Therefore, it is necessary to elucidate the biological status of PRMs to develop alternative PRM control strategies. Quantitative polymerase chain reaction (qPCR) allows analysis of the biological status at the transcript level. However, reference genes are preferable for accurate comparison of expression level changes given the large variation in the quality of the PRM samples collected in each farm. This study aimed to identify candidate reference genes with stable expression levels in the different blood feeding states and life stages of PRMs. First, we selected candidates based on the following criteria: sufficient expression intensity and no significant expression difference between fed and starved states. We selected and characterized seven candidate reference genes. Among them, we evaluated the gene expression stability between the starved and fed states using RefFinder; moreover, we compared their expression levels in each life-stage and identified two reference genes, Elongation factor 1-alpha (ELF1A)-like and apolipophorins-like. Finally, we evaluated the utility of the candidates as reference genes, and the use of ELF1A-like and apolipophorins-like successfully normalized ATP synthase subunit g -like gene expression. Thus, ELF1A-like and apolipophorins-like could be suitable reference genes in PRMs.

摘要

鸡皮刺螨(PRMs,鸡皮刺螨属)是有害的体外寄生虫,会影响养殖鸡,并在全球家禽业造成严重经济损失。杀螨剂用于控制鸡皮刺螨;然而,一些鸡皮刺螨已产生抗杀螨剂特性,这表明需要采用不同方法来控制鸡皮刺螨。因此,有必要阐明鸡皮刺螨的生物学状态,以制定替代的鸡皮刺螨控制策略。定量聚合酶链反应(qPCR)可在转录水平分析生物学状态。然而,鉴于每个养殖场采集的鸡皮刺螨样本质量差异很大,为准确比较表达水平变化,使用参考基因更为可取。本研究旨在鉴定在鸡皮刺螨不同吸血状态和生命阶段具有稳定表达水平的候选参考基因。首先,我们根据以下标准选择候选基因:表达强度足够,在饱血和饥饿状态之间无显著表达差异。我们选择并鉴定了七个候选参考基因。其中,我们使用RefFinder评估了饥饿和饱血状态之间的基因表达稳定性;此外,我们比较了它们在每个生命阶段的表达水平,并鉴定出两个参考基因,类延伸因子1-α(ELF1A)和类载脂蛋白。最后,我们评估了这些候选基因作为参考基因的实用性,使用类ELF1A和类载脂蛋白成功地标准化了类ATP合酶亚基g基因的表达。因此,类ELF1A和类载脂蛋白可能是鸡皮刺螨合适的参考基因。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fa14/8111338/b18fb9baefc0/jvms-83-558-g001.jpg

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