Validation of RT-qPCR Approaches to Monitor<i>Pseudomonas syringae</i>Gene Expression During Infection and Exposure to Pattern-Triggered Immunity

Smith, Amy M.; Lovelace, Amelia H.; Kvitko, Brian H.

doi:10.1094/mpmi-11-17-0270-ta

Cited by 23 publications

(14 citation statements)

References 45 publications

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“…The last temperature was determined by a melting curve analysis. The amplification efficiency was estimated by the serial-dilution method (eight dilutions) [9]. The copy number of target sequences was estimated by optical density, according to the exact molar mass derived from the DNA sequence [10].…”

Section: Methodsmentioning

confidence: 99%

One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection

Meza-Robles

Barajas-Saucedo

Tiburcio-Jimenez

et al. 2020

J Infect Dev Ctries

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Introduction: Due to the coronavirus pandemic, identifying the infected individuals has become key to limiting its spread. Virus nucleic acid real-time RT-PCR testing has become the current standard diagnostic method but high demand could lead to shortages. Therefore, we propose a detection strategy using a one-step nested RT-PCR. Methodology: The nucleotide region in the ORF1ab gene that has the greatest differences between the human coronavirus and the bat coronavirus was selected. Primers were designed after that sequence. All diagnostic primers are species-specific since the 3´ end of the sequence differs from that of other species. A primer set also creates a synthetic positive control. Amplified products were seen in a 2.5% agarose gel, as well as in an SYBR Green-Based Real-Time RT-PCR. Results: Amplification was achieved for the positive control and specific regions in both techniques. Conclusions: This new technique is flexible and easy to implement. It does not require a real-time thermocycler and can be interpreted in agarose gels, as well as adapted to quantify the viral genome. It has the advantage that if the coronavirus mutates in one of the key amplification nucleotides, at least one pair can still amplify, thanks to the four diagnostic primers.

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Section: Methodsmentioning

confidence: 99%

One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection

Meza-Robles

Barajas-Saucedo

Tiburcio-Jimenez

et al. 2020

J Infect Dev Ctries

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“…Then, based on the results of RNA-Seq (described above) we selected the genes that had similar expression levels across all culture conditions as reference genes for each bacterium. According to the method of Smith et al (2018 ), RT-qPCR results were processed using the following equation: …”

Section: Methodsmentioning

confidence: 99%

Phytotoxin synthesis genes and type III effector genes ofPseudomonas syringaepv.actinidiaebiovar 6 are regulated by culture conditions

Hirose

Ishiga

Fujikawa

2020

Preprint

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The kiwifruit bacterial canker (Pseudomonas syringae pv. actinidiae; Psa) causes severe damage to kiwifruit production worldwide. Psa biovar 6 (Psa6), which was isolated in Japan in 2015, produces two types of phytotoxins: coronatine and phaseolotoxin. To date, no other phytopathogenic bacteria are known to produce two phytotoxins. To elucidate the unique virulence of Psa6, we performed transcriptomic analysis of phytotoxin synthesis genes and type III effector genes in in vitro cultivation using various media. The genes related to phytotoxin synthesis and effectors of Psa6 were strictly regulated in the coronatine-inducing mediums (HS and HSC); 14 of 23 effector genes and a hrpL sigma factor gene were induced at 3 h after transferring to the media (early-inducible genes), and phytotoxin synthesis genes such as argD of phaseolotoxin and cfl of coronatine were induced at 6 and 12 h after transferring to the media (late-inducible genes). In contrast, induction of these genes was not observed in the hrp-inducing medium. Next, to examine whether the changes in gene expression in different media is specific to Psa6, we investigated gene expression in other related bacteria. For Psa biovar 1 (Psa1), biovar 3 (Psa3), and P. s. pv. glycinea (Psg), no clear trends were observed in expression behavior across various culture media and incubation times. Therefore, Psa6 seems to exert its virulence efficiently by using two phytotoxins and effectors according to environmental changes. This is not seen in other biovars and pathovars, so it is thought that Psa6 has acquired its own balance of virulence.

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“…(Hellemans et al 2007;Pfaffl 2001) that included in the denominator the geometric mean of reference genes (RG). Normalized Relative Quantity (NRQ) equation described by Smith et al (2018) is presented below:…”

Section: Maize Gene Expression During Rz2ms9 Interactionmentioning

confidence: 99%

New insights into Plant Growth Promoting Rhizobacterium >i<Bacillus thuringiensis>/i< RZ2MS9 biology: entomopathogenic activity and molecular interaction with >i<Zea mays>/i< L.

Longatto¹

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pela oportunidade de realizar o curso de Doutorado. À minha orientadora profª Drª Maria Carolina Quecine Verdi pela inspiração, acolhimento, compreensão, pragmatismo, bons conselhos e, sobretudo, por acreditar em nosso trabalho, mesmo em meio às mudanças. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) processo 140590/2017 e ao Programa de Aperfeiçoamento do Ensino (PAE-USP) pelas bolsas concedidas. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Código de Financiamento 001. Ao professor Dr. João Lucio de Azevedo pela agradável convivência, sabedoria, entusiasmo e bom humor. Ao professor Dr. Roberto Fristche Neto do Laboratório de Plantas Alógamas da ESALQ pela disponibilização das sementes de milho utilizadas nos experimentos de casa de vegetação e da estufa utilizada para secagem de amostras vegetais. À professora Dra. Claudia Barros Monteiro Vitorello pelo apoio e aconselhamento em momentos importantes e por ter disponibilizado equipamento para realização das análises de expressão gênica. Ao professor Dr. José Roberto Postali Parra e a querida técnica Sra Neide Graciano Zério e demais colegas do Laboratório de Biologia de Insetos da ESALQ por terem nos recebido tão bem e fornecido todo suporte para realização dos testes entomológicos. À professora Dra. Maria Lúcia Carneiro Vieira, do Laboratório de Biologia Celular e Molecular de Plantas da ESALQ, pela disponibilização das estufas utilizadas para experimento de casa de vegetação e para preparo de vidraria para extrações de RNA. Aos professores Dr. Fábio Tebaldi Nogueira, Dr. Marcio Castro Silva-Filho e minha orientadora, e colegas Carolina, Filipe, Gleicy, Paula, Renata e Raphael, pelos ensinamentos, conselhos, apoio e atitudes apreendidos durante os dois Estágios Supervisionados em Docência realizados na ESALQ. Ao professor Dr. Antonio Vargas de Oliveira Figueira e Dra. Flavia Bento pelo aprendizado, convivência, respeito e conselhos que levarei comigo da etapa vivida no CENA.

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Validation of RT-qPCR Approaches to MonitorPseudomonas syringaeGene Expression During Infection and Exposure to Pattern-Triggered Immunity

Cited by 23 publications

References 45 publications

One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection

One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection

Phytotoxin synthesis genes and type III effector genes ofPseudomonas syringaepv.actinidiaebiovar 6 are regulated by culture conditions

New insights into Plant Growth Promoting Rhizobacterium >i<Bacillus thuringiensis>/i< RZ2MS9 biology: entomopathogenic activity and molecular interaction with >i<Zea mays>/i< L.

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