Detection and Quantification of Phialophora gregata in Soybean and Soil Samples with a Quantitative, Real-Time PCR Assay

Brown stem rot of soybean, caused by the soilborne fungus Phialophora gregata, is a common and widespread disease of soybean (Glycine max) in the midwestern United States. This pathogen is challenging to study due to a long latent period and slow growth. A TaqMan probe-based quantitative, real-time polymerase chain reaction (qPCR) assay was developed for sensitive and specific detection and quantification of genotypes A and B of P. gregata in plant and soil samples. It is sensitive with detection limits of 50 fg of pure genomic DNA, 100 copies of the target DNA sequence, and approximately 400 conidia. The qPCR assay is approximately 1,000 times more sensitive in detecting DNA and conidia of P. gregata, and is more rapid and less sensitive to PCR inhibitors from soybean stems than a standard PCR (sPCR) assay. Using this single-step qPCR assay, low levels of infection were detected in soybean stems at least 1 to 2 weeks prior to symptom development and before P. gregata was detected with sPCR. This assay also was used to detect the pathogen in field-grown plants and in naturally infested field soils. This new qPCR assay is a powerful tool for rapid, specific, and sensitive detection, diagnosis, and quantification of P. gregata in plants and soil, and for advancing studies of the ecology of P. gregata and its interactions with host plants.

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Real-Time PCR Assays for the Quantification of Phialophora gregata f. sp. sojae IGS Genotypes A and B

Phytopathology ◽

10.1094/phyto-99-9-1008 ◽

2009 ◽

Vol 99 (9) ◽

pp. 1008-1014 ◽

Cited By ~ 6

Author(s):

T. J. Hughes ◽

Z. K. Atallah ◽

C. R. Grau

Keyword(s):

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Intergenic Spacer ◽

Qpcr Assay ◽

Soil Dna ◽

Intergenic Spacer Region ◽

Brown Stem Rot ◽

Phialophora Gregata ◽

Probe Set ◽

Genotype A

Populations of Phialophora gregata f. sp. sojae, the causal agent of brown stem rot (BSR) of soybean, consist of two genotypes, designated A and B. These genotypes are differentiated by an insertion or deletion in the intergenic spacer region (IGS) of ribosomal DNA. The two genotypes differ in the type and severity of symptoms they cause and have displayed preferential host colonization. Methods to quantify populations of P. gregata f. sp. sojae and to distinguish between the two genotypes are essential to understanding this host–pathogen interaction and to improving control of BSR. A real-time, quantitative polymerase chain reaction (qPCR) assay was developed for the specific detection and quantification of P. gregata f. sp. sojae genotype A. This assay is specific to P. gregata f. sp. sojae genotype A, sensitive to 50 fg of DNA, and unaffected by the presence of soybean or soil DNA. When the P. gregata f. sp. sojae genotype A-specific primer/probe set is used in a multiplex qPCR assay with a previously developed primer/probe set which indiscriminately amplifies both genotypes, the quantity of P. gregata f. sp. sojae genotype B can be indirectly determined. This multiplex assay provides a rapid and robust method for studying both the population size and genetic structure of P. gregata f. sp. sojae in its soybean host and in the soil.

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Development of a TaqMan-based real-time PCR assay for detection and quantification of Pythium aphanidermatum in plant and soil samples

New Zealand Journal of Crop and Horticultural Science ◽

10.1080/01140671.2020.1775659 ◽

2020 ◽

Vol 48 (4) ◽

pp. 244-256

Author(s):

Jia Yu ◽

Heng Xu ◽

Xinyu Lu ◽

Yuee Tian ◽

Hao Peng ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pythium Aphanidermatum ◽

Soil Samples ◽

Pcr Assay ◽

Detection And Quantification

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Real-time reverse transcription polymerase chain reaction assay using TaqMan probe for detection and quantification of Infectious myonecrosis virus (IMNV)

Aquaculture ◽

10.1016/j.aquaculture.2006.11.030 ◽

2007 ◽

Vol 264 (1-4) ◽

pp. 9-15 ◽

Cited By ~ 41

Author(s):

Thales P.D. Andrade ◽

Thinnarat Srisuvan ◽

Kathy F.J. Tang ◽

Donald V. Lightner

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Reverse Transcription ◽

Polymerase Chain Reaction Assay ◽

Taqman Probe ◽

Chain Reaction ◽

Infectious Myonecrosis Virus ◽

Polymerase Chain ◽

Detection And Quantification

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Application of quantitative PCR for detection of Mycoplasma suis in blood samples

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/bvip-2014-0082 ◽

2014 ◽

Vol 58 (4) ◽

pp. 533-539

Author(s):

Artur Jabłoński ◽

Dominika Borowska ◽

Sylwia Zębek ◽

Andrzej Kowalczyk ◽

Arkadiusz Dors ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Real Time Pcr ◽

Quantitative Method ◽

Taqman Probe ◽

Blood Samples ◽

Mycoplasma Suis ◽

Coefficients Of Variation ◽

Pcr Method ◽

Detection And Quantification

Abstract The aim of the study was to develop and validate a real-time PCR method, using a TaqMan probe, for quantification of Mycoplasma suis in porcine blood. No PCR signals with closely related non-haemotrophic mycoplasmas were obtained. The detection limit of PCR for plasmid combined with blood DNA was determined to be 103/reaction (5 μL of DNA) (1.2x105 target copies in 1 mL of blood). The linearity of real-time PCR (near 1) indicates its use as a quantitative method. Real-time and quantitative PCR were sensitive and specific for the detection and quantification of M. suis in the blood of animals with acute and chronic form of eperythrozoonosis. Developed quantitative PCR cannot be used to detect carrier animals with a small amount of M. suis in their blood. The validity of real-time PCR used in the studies was confirmed by the low inter- and intra-assay coefficients of variation. This fact confirms the applicability of the assay in other laboratories.

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Molecular detection and quantification of Xanthomonas albilineans in juice from symptomless sugarcane stalks using a real-time quantitative PCR assay

Plant Disease ◽

10.1094/pdis-03-21-0468-re ◽

2021 ◽

Author(s):

Yang Shi ◽

Jian-Ying Zhao ◽

Jing-Ru Zhou ◽

Mbuya Sylvain Ntambo ◽

Peng-Yuan Xu ◽

...

Keyword(s):

Qpcr Assay ◽

Taqman Probe ◽

Bacterial Suspension ◽

Bacterial Disease ◽

Conventional Pcr ◽

Pcr Assay ◽

Leaf Scald ◽

Sugarcane Production ◽

Xanthomonas Albilineans ◽

Detection And Quantification

Leaf scald, a bacterial disease caused by Xanthomonas albilineans (Ashby) Dowson, is a major limiting factor for sugarcane production worldwide. Accurate identification and quantification of X. albilineans is a prerequisite for successful management of this disease. A very sensitive and robust qPCR assay was developed in this study for detection and quantification of X. albilineans using TaqMan probe and primers targeting a putative adenosine triphosphate-binding cassette (ABC) transporter gene (abc). The novel qPCR assay was highly specific to the 43 tested X. albilineans strains belonging to different pulsed-field gel electrophoresis (PFGE) groups. The detection thresholds were 100 copies/µL of plasmid DNA, 100 fg/µL of bacterial genomic DNA, and 100 CFU/ml of bacterial suspension prepared from pure culture. This qPCR assay was 100 times more sensitive than a conventional PCR assay. The pathogen was detected by qPCR in 75.1% (410/546) symptomless stalk samples, whereas only 28.4% (155/546) samples tested positive by conventional PCR. Based on qPCR data, population densities of X. albilineans in symptomless stalks of the same varieties differed between two sugarcane production areas in China, Beihai (Guangxi province) and Zhanjiang (Guangdong province), and no significant correlation between these populations was identified. Furthermore, no relationship was found between these populations of the pathogen in asymptomatic stalks and the resistance level of the sugarcane varieties to leaf scald. The newly developed qPCR assay proved to be highly sensitive and reliable for the detection and quantification of X. albilineans in sugarcane stalks.

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Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR

Plant Disease ◽

10.1094/pdis-02-18-0310-re ◽

2018 ◽

Vol 102 (12) ◽

pp. 2487-2493 ◽

Cited By ~ 2

Author(s):

Jeremiah K.S. Dung ◽

Jeness C. Scott ◽

Qunkang Cheng ◽

Stephen C. Alderman ◽

Navneet Kaur ◽

...

Keyword(s):

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Qpcr Assay ◽

Management Approach ◽

Conidial Suspension ◽

Claviceps Purpurea ◽

Spore Trap ◽

Grass Seed ◽

Wide Range ◽

Detection And Quantification

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = –0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = –0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = –0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.

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Rapid and Quantitative Detection ofLeifsonia xylisubsp.xyliin Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

BioMed Research International ◽

10.1155/2016/2681816 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Hua-Ying Fu ◽

Sheng-Ren Sun ◽

Jin-Da Wang ◽

Kashif Ahmad ◽

Heng-Bo Wang ◽

...

Keyword(s):

Real Time ◽

Disease Incidence ◽

Quantitative Polymerase Chain Reaction ◽

Field Trials ◽

Diagnostic Methods ◽

Qpcr Assay ◽

Taqman Probe ◽

Quantitative Detection ◽

Conventional Pcr ◽

Sugarcane Stalk

Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agentLeifsonia xylisubsp.xyli(Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification ofLxxdetection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting thePart1gene ofLxxwere used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg ofLxxgenomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods forLxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

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Development of real-time PCR assay using TaqMan probe for detection and quantification of Rosellinia necatrix in plant and soil

Journal of General Plant Pathology ◽

10.1007/s10327-012-0366-x ◽

2012 ◽

Vol 78 (2) ◽

pp. 115-120 ◽

Cited By ~ 6

Author(s):

Masahiro Shishido ◽

Itsuki Kubota ◽

Hitoshi Nakamura

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Taqman Probe ◽

Rosellinia Necatrix ◽

Pcr Assay ◽

Detection And Quantification

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Quantification of Paratrichodorus allius in DNA extracted from soil using TaqMan Probe and SYBR Green real-time PCR assays

Nematology ◽

10.1163/15685411-00003101 ◽

2017 ◽

Vol 19 (8) ◽

pp. 987-1001 ◽

Cited By ~ 10

Author(s):

Danqiong Huang ◽

Guiping Yan ◽

Neil Gudmestad ◽

Andrea Skantar

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Diagnostic Method ◽

Potato Industry ◽

Soil Samples ◽

Sybr Green ◽

Taqman Probe ◽

Field Soil ◽

Soil Dna ◽

Pcr Assays

The ectoparasitic stubby root nematode,Paratrichodorus allius, transmits tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. A diagnostic method for direct quantification ofP. alliusfrom soil DNA using TaqMan probe and SYBR Green real-time PCR assays was developed to assist the potato industry in management of this important vector. Specificity of primers/probe designed from the internal transcribed spacer of ribosomal DNA ofP. alliuswas demonstrated byin silicoanalysis and experimental PCR tests with no cross reactions using non-target nematode species and nematode communities. The SYBR Green method was more sensitive than the TaqMan probe method during detection using serial diluted DNA templates. Standard curves were generated from serial dilutions of DNA extracted from autoclaved soil with artificially inoculatedP. alliusindividuals and were validated by high correlations between the numbers of target nematodes quantified by the assays and added to the soil. Moreover, the numbers ofP. alliusdetermined by the real-time PCR assays and estimated by the microscopic method in 17 field soil samples presented positive correlation relationships (). Although the quantification using TaqMan probe overestimated the target nematodes compared to using SYBR Green in eight out of ten field soil samples, results of the two methods correlated well (). This is the first report ofP. alliusquantification from soil DNA extracts using real-time PCR, providing a rapid and sensitive diagnostic method obviating time-consuming manual nematode extraction from soil and microscopic identification and quantification.

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