Ilakkiya Thirugnanasambandam, Tara Vucurevich, Tara Shelton, Nat N. V. Kav, Claudia Sheedy, André Laroche and Jonathan K. Challis*,
{"title":"评估用于检测小麦空气传播真菌病原体的实时 PCR 技术：DNA 提取对孢子定量的作用","authors":"Ilakkiya Thirugnanasambandam, Tara Vucurevich, Tara Shelton, Nat N. V. Kav, Claudia Sheedy, André Laroche and Jonathan K. Challis*, ","doi":"10.1021/acsagscitech.4c0038210.1021/acsagscitech.4c00382","DOIUrl":null,"url":null,"abstract":"<p >Our research focused on developing a highly sensitive whole-spore real-time immuno-PCR (RT-iPCR) assay for the detection of three wheat fungal pathogens: <i>Pyrenophora tritici-repentis</i> (<i>Ptr</i>), <i>Fusarium graminearum</i> (<i>Fg</i>), and <i>Puccinia striiformis</i> forma specialis (f. sp.) <i>tritici</i> (<i>Pst</i>). RT-iPCR measurements were compared to more well-established quantitative PCR (qPCR) assays to compare their performance. While specificity remained a challenge for RT-iPCR, the direct spore measurements negate the need for DNA extraction, making RT-iPCR a potentially valuable technique that warrants further research. An alternative approach was developed to determine DNA extraction efficiency and quantification of spore numbers by qPCR, which is currently a methodological gap in most qPCR spore measurements. DNA extraction efficiency determined for <i>Fg, Pst</i>, and <i>Ptr</i> spores were 5.0 ± 0.1, 7.0 ± 0.4, and 290 ± 36%, respectively, demonstrating important implications for the accuracy of these techniques when DNA recovery is not considered.</p>","PeriodicalId":93846,"journal":{"name":"ACS agricultural science & technology","volume":null,"pages":null},"PeriodicalIF":2.3000,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsagscitech.4c00382","citationCount":"0","resultStr":"{\"title\":\"Assessment of Real-Time PCR Techniques for the Detection of Airborne Fungal Pathogens of Wheat: Role of DNA Extraction on Spore Quantification\",\"authors\":\"Ilakkiya Thirugnanasambandam, Tara Vucurevich, Tara Shelton, Nat N. V. Kav, Claudia Sheedy, André Laroche and Jonathan K. Challis*, \",\"doi\":\"10.1021/acsagscitech.4c0038210.1021/acsagscitech.4c00382\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >Our research focused on developing a highly sensitive whole-spore real-time immuno-PCR (RT-iPCR) assay for the detection of three wheat fungal pathogens: <i>Pyrenophora tritici-repentis</i> (<i>Ptr</i>), <i>Fusarium graminearum</i> (<i>Fg</i>), and <i>Puccinia striiformis</i> forma specialis (f. sp.) <i>tritici</i> (<i>Pst</i>). RT-iPCR measurements were compared to more well-established quantitative PCR (qPCR) assays to compare their performance. While specificity remained a challenge for RT-iPCR, the direct spore measurements negate the need for DNA extraction, making RT-iPCR a potentially valuable technique that warrants further research. An alternative approach was developed to determine DNA extraction efficiency and quantification of spore numbers by qPCR, which is currently a methodological gap in most qPCR spore measurements. DNA extraction efficiency determined for <i>Fg, Pst</i>, and <i>Ptr</i> spores were 5.0 ± 0.1, 7.0 ± 0.4, and 290 ± 36%, respectively, demonstrating important implications for the accuracy of these techniques when DNA recovery is not considered.</p>\",\"PeriodicalId\":93846,\"journal\":{\"name\":\"ACS agricultural science & technology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.3000,\"publicationDate\":\"2024-09-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://pubs.acs.org/doi/epdf/10.1021/acsagscitech.4c00382\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ACS agricultural science & technology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://pubs.acs.org/doi/10.1021/acsagscitech.4c00382\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"AGRICULTURE, MULTIDISCIPLINARY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS agricultural science & technology","FirstCategoryId":"1085","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acsagscitech.4c00382","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"AGRICULTURE, MULTIDISCIPLINARY","Score":null,"Total":0}
Assessment of Real-Time PCR Techniques for the Detection of Airborne Fungal Pathogens of Wheat: Role of DNA Extraction on Spore Quantification
Our research focused on developing a highly sensitive whole-spore real-time immuno-PCR (RT-iPCR) assay for the detection of three wheat fungal pathogens: Pyrenophora tritici-repentis (Ptr), Fusarium graminearum (Fg), and Puccinia striiformis forma specialis (f. sp.) tritici (Pst). RT-iPCR measurements were compared to more well-established quantitative PCR (qPCR) assays to compare their performance. While specificity remained a challenge for RT-iPCR, the direct spore measurements negate the need for DNA extraction, making RT-iPCR a potentially valuable technique that warrants further research. An alternative approach was developed to determine DNA extraction efficiency and quantification of spore numbers by qPCR, which is currently a methodological gap in most qPCR spore measurements. DNA extraction efficiency determined for Fg, Pst, and Ptr spores were 5.0 ± 0.1, 7.0 ± 0.4, and 290 ± 36%, respectively, demonstrating important implications for the accuracy of these techniques when DNA recovery is not considered.
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