Multiplex Real-Time Quantitative PCR to Detect and Quantify Verticillium dahliae Colonization in Potato Lines that Differ in Response to Verticillium Wilt

Potato early dying (PED), also known as Verticillium wilt, caused by Verticillium dahliae, is a seasonal yield-limiting disease of potato worldwide, and PED-resistant cultivars currently represent only a small percentage of potato production. In this study, we developed a real-time quantitative polymerase chain reaction (Q-PCR) approach to detect and quantify V. dahliae. The efficiency of the designed primer pair VertBt-F/VertBt-R, derived from the sequence of the β-tubulin gene, was greater than 95% in monoplex Q-PCR and duplex (using Plexor technology) procedures with primers PotAct-F/PotAct-R, obtained from the sequence of the actin gene, designed for potato. As few as 148 fg of V. dahliae DNA were detected and quantified, which is equivalent to five nuclei. Q-PCR detected V. dahliae in naturally infected air-dried potato stems and fresh stems of inoculated plants. Spearman correlations indicated a high correlation (upward of 80%) between V. dahliae quantifications using Q-PCR and the currently used plating assays. Moreover, Q-PCR substantially reduced the variability compared with that observed in the plating assay, and allowed for the detection of V. dahliae in 10% of stem samples found to be pathogen free on the culture medium. The described Q-PCR approach should provide breeders with a more sensitive and less variable alternative to time-consuming plating assays to distinguish response of breeding lines to colonization by V. dahliae.

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Diagnosis and follow-up of Bartonella henselae infection in the spleen of an immunocompetent patient by real-time quantitative PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.050617-0 ◽

2013 ◽

Vol 62 (7) ◽

pp. 1081-1085 ◽

Cited By ~ 6

Author(s):

Maria Carla Liberto ◽

Giovanni Matera ◽

Angelo G. Lamberti ◽

Angela Quirino ◽

Giorgio S. Barreca ◽

...

Keyword(s):

Real Time ◽

Bartonella Henselae ◽

Quantitative Polymerase Chain Reaction ◽

Immunocompetent Patient ◽

Chain Reaction ◽

Real Time Quantitative Pcr ◽

Novel Method ◽

Polymerase Chain ◽

Immunocompetent Adults

Systemic Bartonella henselae infections are unusual in immunocompetent adults. However, here we report one such case of bartonellosis in a 34-year-old patient, who presented with fever and multinodular splenomegaly. We also describe a novel method of identifying Bartonella henselae by real-time quantitative polymerase chain reaction and sequencing of amplified products. This could prevent splenic bartonellosis being mistaken for lymphoma and thereby avert unnecessary splenectomy.

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Real-time quantitative PCR detection ofEscherichia coliO157:H7

Chinese Journal of Agricultural Biotechnology ◽

10.1017/s1479236207001349 ◽

2007 ◽

Vol 4 (1) ◽

pp. 15-19 ◽

Cited By ~ 4

Author(s):

Chen Si ◽

Huang Kun-Lun ◽

Xu Wen-Tao ◽

Li Yuan ◽

Luo Yun-Bo

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Polymerase Chain Reaction ◽

Amplification Product ◽

Chain Reaction ◽

Standard Curve ◽

Real Time Quantitative Pcr ◽

Pcr Method ◽

Polymerase Chain ◽

Dissociation Curves

AbstractA rapid and accurate real-time quantitative polymerase chain reaction (real-time PCR) method with SYBR Green I was established for detectingEscherichia coliO157:H7. A pair of primers were designed to amplify theeaegene. The dissociation curves showed that the amplification product was very specific. The optimal conditions and standard curve were established. The result indicated that real-time PCR was 1000 times more sensitive than ordinary PCR.

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A Real-Time PCR Assay for Detection and Quantification of Verticillium dahliae in Spinach Seed

Phytopathology ◽

10.1094/phyto-10-11-0280 ◽

2012 ◽

Vol 102 (4) ◽

pp. 443-451 ◽

Cited By ~ 36

Author(s):

Dechassa Duressa ◽

Gilda Rauscher ◽

Steven T. Koike ◽

Beiquan Mou ◽

Ryan J. Hayes ◽

...

Keyword(s):

Real Time ◽

Verticillium Wilt ◽

Verticillium Dahliae ◽

Real Time Pcr ◽

The United States ◽

Infected Seed ◽

Polymerase Chain ◽

Reliable Quantification ◽

Pathogen Dna ◽

Detection And Quantification

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.

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Assessment of Resistance in Potato Cultivars to Verticillium Wilt Caused by Verticillium dahliae and Verticillium nonalfalfae

Plant Disease ◽

10.1094/pdis-10-18-1815-re ◽

2019 ◽

Vol 103 (6) ◽

pp. 1357-1362

Author(s):

Haiyuan Li ◽

Zhipeng Wang ◽

Xiaoping Hu ◽

Wenjing Shang ◽

Ruiqing Shen ◽

...

Keyword(s):

Verticillium Wilt ◽

Verticillium Dahliae ◽

Potato Production ◽

Potato Cultivars ◽

Progress Curve ◽

Early Dying Disease ◽

Verticillium Nonalfalfae ◽

Early Dying ◽

Cultivar Responses ◽

Production Areas

Verticillium wilt caused by Verticillium spp., also called potato early dying disease, is one of the most serious soilborne diseases affecting potato production in China. The disease has been expanding into most potato production areas over the past few years. Information on host resistance against Verticillium wilt among the potato cultivars in China is scarce, but it is critical for sustainable management of the disease. This study, therefore, evaluated 30 commercially popular potato cultivars against Verticillium dahliae strain Vdp83 and Verticillium nonalfalfae strain Vnp24, two well-characterized strains causing Verticillium wilt of potato in China. Both strains were isolated from diseased potato plants, and they were previously proven to be highly virulent. Ten plants of each cultivar were inoculated with the V. dahliae strain and incubated on greenhouse benches. Symptoms were rated at weekly intervals, and the relative area under the disease progress curve was calculated. The experiment was repeated once, and nonparametric analysis was used to calculate the relative marginal effects and the corresponding confidence intervals. Five resistant cultivars and four susceptible cultivars identified from the analyses were then challenged with the V. nonalfalfae strain. Cultivar responses to V. nonalfalfae were like those exhibited against V. dahliae, except for one cultivar. This study showed that resistance among potato cultivars exists in China against Verticillium spp. and that the resistance to V. dahliae identified in potato is also effective against the other Verticillium species that cause Verticillium wilt with a few exceptions. Cultivars identified as resistant to Verticillium wilt can be deployed to manage the disease until the breeding programs develop new cultivars with resistance from the sources identified in this study.

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A Comparison of Real-Time PCR Protocols for the Quantitative Monitoring of Asymptomatic Olive Infections by Verticillium dahliae Pathotypes

Phytopathology ◽

10.1094/phyto-11-12-0312-r ◽

2013 ◽

Vol 103 (10) ◽

pp. 1058-1068 ◽

Cited By ~ 20

Author(s):

D. Gramaje ◽

V. Pérez-Serrano ◽

M. Montes-Borrego ◽

J. A. Navas-Cortés ◽

R. M. Jiménez-Díaz ◽

...

Keyword(s):

Real Time ◽

Verticillium Wilt ◽

Verticillium Dahliae ◽

Target Gene ◽

Quantitative Polymerase Chain Reaction ◽

Intergenic Spacer ◽

In Planta ◽

Development Of Resistance ◽

Planting Material ◽

Detection And Quantification

Early, specific, and accurate in planta detection and quantification of Verticillium dahliae are essential to prevent the spread of Verticillium wilt in olive using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, specificity, and efficiency of eight real-time quantitative polymerase chain reaction protocols published since 2002 for the specific detection and quantification of V. dahliae in various host plant species and in soil, using a background of DNAs extracted from olive roots, stems, and leaves. Results showed that some of those protocols were not specific for V. dahliae or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocols TAQ (based on intergenic spacer ribosomal DNA target gene) and SYBR-4 (based on the β-tubulin 2 target gene) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and stem) tested. Use of TAQ and SYBR-4 protocols allowed accurate quantification of V. dahliae DNA regardless of the background DNA, with a detection limit being fixed at a cycle threshold of 36 (≈18 fg for SYBR-4 and 15 fg for TAQ) of V. dahliae. The amount of DNA from defoliating (D) and nondefoliating (ND) V. dahliae pathotypes was monitored in Verticillium wilt-resistant ‘Frantoio’ olive using the TAQ and SYBR-4 protocols. In the infection bioassay, higher amounts of D V. dahliae DNA were measured in olive stems, whereas the average amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amounts in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days after inoculation). The SYBR-4 and TAQ protocols further enabled detection of V. dahliae in tissues of symptomless plants, suggesting that both techniques can be useful for implementing certification schemes of pathogen-free planting material as well as helpful tools in breeding resistance to V. dahliae in olive.

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Colonization Dynamics and Spatial Progression of Verticillium dahliae in Individual Stems of Two Potato Cultivars with Differing Responses to Potato Early Dying

Plant Disease ◽

10.1094/pdis-91-9-1137 ◽

2007 ◽

Vol 91 (9) ◽

pp. 1137-1141 ◽

Cited By ~ 17

Author(s):

J. Bae ◽

Z. K. Atallah ◽

S. H. Jansky ◽

D. I. Rouse ◽

W. R. Stevenson

Keyword(s):

Verticillium Dahliae ◽

Potato Cultivars ◽

Conidial Suspension ◽

Potato Early Dying ◽

Ranger Russet ◽

Russet Norkotah ◽

Colonization Dynamics ◽

Polymerase Chain ◽

Early Dying ◽

Moderately Resistant

Potato early dying (PED), caused by Verticillium dahliae, is a chronic yield-limiting disease of potato (Solanum tuberosum). In this study, we describe the colonization dynamics of V. dahliae in two potato cultivars with varying responses to PED. We utilized a quantitative real-time polymerase chain reaction (Q-PCR) assay to assess the colonization and spatial progression of V. dahliae in cvs. Ranger Russet (moderately resistant) and Russet Norkotah (highly susceptible). Ninety plants per cultivar were inoculated with a conidial suspension in the greenhouse. Every 2 weeks until week 10, we collected basal samples from 15 plants, and repeatedly sampled the growing apices of another 15 plants. The mean infection coefficient (IC) values in the basal and apical samples were significantly lower in cv. Ranger Russet at all five sampling dates. The pathogen was detected in basal samples of both cultivars by week 2, and in apical samples of cv. Russet Norkotah at week 4 and of cv. Ranger Russet at week 6. Colonization of cv. Russet Norkotah consistently increased in apical and basal samples during the 10 weeks, while it plateaued after week 6 in cv. Ranger Russet. Differences in response to PED appear associated with the speed of colonization and the establishment of a higher population density by V. dahliae in the plant.

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Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

Virology Journal ◽

10.1186/s12985-020-01467-y ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Yang Zhang ◽

Chunyang Dai ◽

Huiyan Wang ◽

Yong Gao ◽

Tuantuan Li ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Clinical Samples ◽

Chain Reaction ◽

Pcr Assay ◽

Qrt Pcr ◽

Highly Sensitive ◽

One Step ◽

Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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