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

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
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.

scholarly journals Detection and Quantification of Pythium tracheiphilum in Soil by Multiplex Real-Time qPCR

Plant Disease ◽  
2019 ◽  
Vol 103 (3) ◽  
pp. 475-483 ◽  
Author(s):  
Hervé Van der Heyden ◽  
Thérèse Wallon ◽  
C. André Lévesque ◽  
Odile Carisse
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Disease Incidence ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Qpcr Assay ◽  
Inoculum Concentration ◽  
Soil P ◽  
Dry Soil ◽  
Detection And Quantification

In Canada, head lettuce (Lactuca sativa capitata) is extensively produced in the muck soils of southwestern Québec. However, yields are increasingly affected by various soilborne pathogens, including Pythium spp., which cause wilt and damping off. In a survey conducted in Québec muck soils in 2010 and 2011, Pythium tracheiphilum Matta was identified as the predominant Pythium sp. in the root of head lettuce showing Pythium stunt symptoms. Therefore, to improve risk assessment and help further understanding of disease epidemiology, a specific and sensitive real-time quantitative polymerase chain reaction (qPCR) assay based on TaqMan-minor groove binder (MGB) technology was developed for P. tracheiphilum. The PCR primers along with a TaqMan-MGB probe were designed from the ribosomal internal transcribed spacer 2 region. A 100-bp product was amplified by PCR from all P. tracheiphilum isolates tested while no PCR product was obtained from 38 other Pythium spp. or from a selection of additional lettuce pathogens tested. In addition to P. tracheiphilum, the assay was multiplexed with an internal control allowing for the individual validation of each PCR. In artificially infested soils, the sensitivity of the qPCR assay was established as 10 oospores/g of dry soil. P. tracheiphilum was not detected in soils in which lettuce has never been grown; however, inoculum ranged from 0 to more than 200,000 oospores/g of dry soil in commercial lettuce fields. Also, disease incidence was positively correlated with inoculum concentration (r = 0.764). The results suggest that inoculum concentration should be considered when making Pythium stunt management decisions. The developed qPCR assay will facilitate reliable detection and quantification of P. tracheiphilum from field soil.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

scholarly journals Conventional PCR Detection and Real-Time PCR Quantification of Reniform Nematodes

Plant Disease ◽  
2012 ◽  
Vol 96 (12) ◽  
pp. 1757-1762 ◽  
Author(s):  
Ronald J. Sayler ◽  
Courtney Walker ◽  
Fiona Goggin ◽  
Paula Agudelo ◽  
Terrence Kirkpatrick
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Its Region ◽  
Taqman Probe ◽  
Reniform Nematode ◽  
Rotylenchulus Reniformis ◽  
Conventional Pcr ◽  
Diagnostic Assays ◽  
Reniform Nematodes

Reniform nematode (Rotylenchulus reniformis) is a relatively recent introduction into the continental United States that can cause major yield losses on a variety of important crops including cotton and soybeans. DNA sequences from the internal transcribed spacer (ITS) region of this nematode were used to design primers for conventional and real-time PCR, as well as a TaqMan probe. These primers amplified DNA of reniform nematode isolates from a wide geographic range but did not detect genetically related species or other pathogenic nematodes found in production fields including Meloidogyne incognita and Heterodera glycines. Both SYBR green and TaqMan assays reliably quantified as little as 100 fg of reniform nematode DNA, and could be used to quantify as few as five reniform nematodes. An inexpensive and rapid DNA extraction protocol for high throughput diagnostic assays is described.

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

Plant Disease ◽  
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.

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

Plant Disease ◽  
2018 ◽  
Vol 102 (12) ◽  
pp. 2487-2493 ◽  
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.

Midturbinate Swabs Are Comparable to Nasopharyngeal Swabs for Quantitative Detection of Respiratory Syncytial Virus in Infants

2018 ◽  
Vol 8 (6) ◽  
pp. 554-558 ◽  
Author(s):  
Anne J Blaschke ◽  
Matt McKevitt ◽  
Krow Ampofo ◽  
Tammi Lewis ◽  
Hao Chai ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Respiratory Syncytial Virus ◽  
Real Time ◽  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Viral Loads ◽  
Polymerase Chain ◽  
Syncytial Virus

Abstract Nasopharyngeal (NP) swabs are generally used to detect respiratory syncytial virus (RSV) in infants. However, midturbinate (MT) swabs may provide comparable results. In this study, we enrolled hospitalized infants aged &lt;24 months with RSV and collected NP and MT swabs. The resulting viral loads measured by real-time reverse-transcription quantitative polymerase chain reaction were similar. Most parents preferred MT swabs over NP swabs.

Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR

2018 ◽  
Vol 151 ◽  
pp. 191-196 ◽  
Author(s):  
Ya-Mei Liu ◽  
Liang Qiu ◽  
An-Zhi Sheng ◽  
Xiao-Yuan Wan ◽  
Dong-Yuan Cheng ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection Method ◽  
Taqman Probe ◽  
Quantitative Detection ◽  
Enterocytozoon Hepatopenaei
Sign in / Sign up

Export Citation Format

Share Document