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重点植物病原体的高靶向检测:从获得多克隆抗体到酶联免疫吸附测定和侧向流动免疫测定的开发与验证

Highly Targeted Detection of Priority Phytopathogen : From Obtaining Polyclonal Antibodies to Development and Approbation of Enzyme-Linked Immunoassay and Lateral Flow Immunoassay.

作者信息

Safenkova Irina V, Galushka Pavel A, Varitsev Yuri A, Kamionskaya Maria V, Drenova Natalia V, Vasilyeva Anna A, Zherdev Anatoly V, Uskov Alexander I, Dzantiev Boris B

机构信息

A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.

Russian Potato Research Centre, 140051 Kraskovo, Moscow region, Russia.

出版信息

Microorganisms. 2024 Nov 27;12(12):2436. doi: 10.3390/microorganisms12122436.

Abstract

is a bacterial phytopathogen that causes soft and black rot and actively spreads worldwide. Our study is the first development of immunoassays for detecting . We immunized rabbits and obtained serum with an extremely high titer (1:10). Isolated polyclonal antibodies were tested by enzyme-linked immunosorbent assay (ELISA) using 18 closely related strains and 5 non-related bacterial pathogens. No cross-reactivity was found concerning the tested pathogens. The ELISA of was developed in a double-antibody sandwich format with a detection limit of 1.5 × 10 cells/mL. A lateral flow immunoassay (LFIA) for was also developed in a double-antibody sandwich format with a detection limit of 1 × 10 cells/mL. The results of cells testing with LFIA in plant matrix showed a high correlation (R = 0.932) between concentrations of added and revealed cells. When testing potato seed material, ELISA and LFIA confirmed 75 and 66% of positive samples according to real-time PCR, respectively. For negative samples, ELISA showed 84% coincidence, and LFIA coincided with PCR for 89% of samples. Thus, the developed immunoassays can be used to evaluate plant material in poorly equipped conditions or under field testing.

摘要

是一种引起软腐病和黑腐病的细菌性植物病原体,并在全球范围内积极传播。我们的研究是首次开发用于检测的免疫测定法。我们对兔子进行免疫并获得了效价极高(1:10)的血清。使用18种密切相关菌株和5种非相关细菌病原体通过酶联免疫吸附测定(ELISA)对分离的多克隆抗体进行了测试。在所测试的病原体中未发现交叉反应。的ELISA采用双抗体夹心形式开发,检测限为1.5×10个细胞/毫升。用于的侧向流动免疫测定(LFIA)也采用双抗体夹心形式开发,检测限为1×10个细胞/毫升。在植物基质中用LFIA对细胞进行测试的结果表明,添加的细胞浓度与检测到的细胞浓度之间具有高度相关性(R = 0.932)。在检测马铃薯种子材料时,ELISA和LFIA分别根据实时PCR确认了75%和66%的阳性样品。对于阴性样品,ELISA显示出84%的一致性,LFIA与PCR在89%的样品中一致。因此,可以使用所开发的免疫测定法在设备简陋的条件下或现场测试中评估植物材料。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6108/11676143/c701bfa57708/microorganisms-12-02436-g001.jpg

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