scholarly journals Evaluation of Cultivar Resistance to Soybean Cyst Nematode with a Quantitative Polymerase Chain Reaction Assay

Plant Disease ◽  
2012 ◽  
Vol 96 (10) ◽  
pp. 1556-1563 ◽  
Author(s):  
Horacio D. Lopez-Nicora ◽  
James P. Craig ◽  
Xuebiao Gao ◽  
Kris N. Lambert ◽  
Terry L. Niblack
Keyword(s):  
Polymerase Chain Reaction ◽  
Soybean Cyst Nematode ◽  
Quantitative Polymerase Chain Reaction ◽  
Cyst Nematode ◽  
Qpcr Assay ◽  
Distilled Water ◽  
Chain Reaction ◽  
Resistant Cultivars ◽  
Soybean Roots ◽  
Polymerase Chain

Heterodera glycines, the soybean cyst nematode, is a major pathogen of soybean. Effective management of this pathogen is contingent on the use of resistant cultivars; thus, screening for resistant cultivars is essential. The purpose of this research was to develop a method to assess infection of soybean roots by H. glycines with real-time quantitative polymerase chain reaction (qPCR). This method will serve as a prelude to differentiation of resistance levels in soybean cultivars. A reproducible inoculation method was developed by means of a sand column to provide active second-stage juveniles (J2). Two-day-old soybean roots were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours after infestation, the roots were surface-sterilized and genomic DNA (gDNA) was extracted. For the qPCR assay, a primer pair for the single copy gene HgSNO, which codes for a protein involved in the production of vitamin B6, was selected for H. glycines gDNA amplification within soybean roots. Compatible ‘Lee 74’, incompatible ‘Peking’, and cultivars with different levels of resistance to H. glycines were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours postinfestation, infected seedlings were transplanted into pasteurized soil. Subsequently, they were harvested at 1, 7, 10, 14, and 21 days postinfestation for gDNA extraction. With the qPCR assay, the time needed to differentiate highly resistant cultivars from the rest was reduced. Quantification of H. glycines infection by traditional means (numbers of females produced in 30 days) is a time-consuming practice. This qPCR assay has the potential to replace the traditional Female Index-based screening and improve precision in determining infection levels.

scholarly journals Clinical Validation of a Novel Commercial Reverse Transcription–Quantitative Polymerase Chain Reaction Screening Assay for Detection of ALK Translocations and Amplifications in Non–Small Cell Lung Carcinomas

2015 ◽  
Vol 140 (7) ◽  
pp. 690-693 ◽  
Author(s):  
Chunyan Liu ◽  
Kristi Pepper ◽  
Heather Hendrickson ◽  
Philip T. Cagle ◽  
Bryce P. Portier
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Genetic Alterations ◽  
Qpcr Assay ◽  
Egfr Mutations ◽  
Chain Reaction ◽  
Lung Cancers ◽  
Anaplastic Lymphoma ◽  
Polymerase Chain

Context.— EGFR mutations and anaplastic lymphoma kinase (ALK) translocations have significant biologic and therapeutic implications in lung cancers, particularly lung adenocarcinomas. ALK translocations are less frequent compared with EGFR mutations; interestingly, these two abnormalities are most commonly mutually exclusive. The 2013 College of American Pathologists/Association for Molecular Pathology/International Association for the Study of Lung Cancer molecular testing guideline for lung cancers recommend a testing algorithm in which detection of ALK translocations using fluorescence in situ hybridization (FISH) is to be performed following testing for EGFR mutations. Such an algorithm is cost-effective but potentially slows down turnaround time; and as a secondary test, ALK FISH assay may not be completed because it requires the use of additional tissue, and the small biopsies or cytology specimens may have been exhausted in the extraction of nucleic acid for EGFR mutation screening.Objective.—To provide efficient testing of both EGFR and ALK genetic alterations in small biopsies and cytology specimens.Design.—We validated a highly sensitive ALK reverse transcription–quantitative polymerase chain reaction (RT-qPCR) assay as a screening tool for ALK translocations and amplifications.Results.—We performed a retrospective review of cases previously tested by FISH and found that all FISH ALK translocation–positive specimens were RT-qPCR positive, and all FISH ALK translocation–negative cases were RT-qPCR negative (the sensitivity and specificity of the ALK RT-qPCR assay were 100%).Conclusion.—This assay allows rapid identification of ALK alterations, can be performed in conjunction with EGFR testing, and does not require use of valuable additional tumor tissue.

Identification of the full-length Hs1pro-1 coding sequence and preliminary evaluation of soybean cyst nematode resistance in soybean transformed with Hs1pro-1 cDNA

2007 ◽  
Vol 85 (4) ◽  
pp. 437-441 ◽  
Author(s):  
Michael D. McLean ◽  
Gordon J. Hoover ◽  
Bonnie Bancroft ◽  
Amina Makhmoudova ◽  
Shawn M. Clark ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Soybean Cyst Nematode ◽  
Nematode Resistance ◽  
Cyst Nematode ◽  
Full Length ◽  
Preliminary Evaluation ◽  
Chain Reaction ◽  
Coding Sequence ◽  
Scn Resistance ◽  
Polymerase Chain

The Hs1pro-1 gene reportedly confers resistance to the beet cyst nematode in wild beet and sugar beet. Here, we tested the hypothesis that Hs1pro-1 confers resistance in soybean against the soybean cyst nematode (SCN). The full-length Hs1pro-1 coding sequence, which encodes a predicted polypeptide of 490 amino acids, was first acquired then expressed in ‘Westag’ soybean using a constitutive octopine synthase – mannopine synthase promoter. Thirty T0 lines that successfully expressed the Hs1pro-1 gene, as indicated by both polymerase chain reaction and reverse transcriptase – polymerase chain reaction analyses, were generated. Bioassay of the T1 progeny from these lines revealed that only five T0 lines grew normally and exhibited a high degree of SCN resistance. On average, these T1 transgenic progeny were about 70% more resistant to SCN than susceptible control cultivars. These preliminary data suggest that Hs1pro-1 is a promising candidate for genetically engineering SCN resistance in elite, locally adapted soybean cultivars.

One for two: A novel and highly sensitive virulence factor-based quantitative polymerase chain reaction assay for the simultaneous detection of Rodentibacter pneumotropicus and Rodentibacter heylii in environmental sample material

2019 ◽  
Vol 54 (3) ◽  
pp. 239-250
Author(s):  
Stephanie Buchheister ◽  
Florian Roegener ◽  
Nils-Holger Zschemisch ◽  
Steven R. Talbot ◽  
Henrik Christensen ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Virulence Factor ◽  
Environmental Sample ◽  
Quantitative Polymerase Chain Reaction ◽  
Simultaneous Detection ◽  
Qpcr Assay ◽  
Environmental Sampling ◽  
Sample Material ◽  
Chain Reaction ◽  
Polymerase Chain

Hygienic monitoring of laboratory rodents has focused more and more on the analysis of environmental sample material by quantitative polymerase chain reaction (qPCR) assays. This approach requires profound knowledge of specific genetic sequences of the agents to be monitored and the assays need to be permanently adapted to take the latest research into account. [ Pasteurella] pneumotropica was recently reclassified into the new genus Rodentibacter, with Rodentibacter (R.) pneumotropicus and R. heylii as the most commonly detected species in laboratory mouse colonies. This study aimed at the development of a specific qPCR assay for the simultaneous detection of both agents. A novel primer probe set, based on detection of the specific virulence factor‚ ‘inclusion body protein A’ gene ( ibpA), was confirmed by testing the assay on currently described Rodentibacter type species and other Pasteurellaceae. Furthermore, it was validated within four different barrier units and results were compared with the cultural analysis of sentinel mice. The assay was suitable to specifically detect R. pneumotropicus and R. heylii and discriminate them from other murine Rodentibacter spp. In addition, it revealed high sensitivity for the detection of both agents in environmental sampling material including exhaust air dust in individually ventilated cage systems. Altogether, higher pathogen prevalence was detected via qPCR of environmental samples compared with cultural diagnostics of sentinel mice. This study describes a qPCR assay for the simultaneous detection of R. pneumotropicus and R. heylii. This assay was demonstrated to be beneficial during routine health monitoring, especially with regard to environmental sampling strategies.

scholarly journals Validation of a Triplex Quantitative Polymerase Chain Reaction Assay for Detection and Quantification of Traditional Protein Sources, Pisum sativum L. and Glycine max (L.) Merr., in Protein Powder Mixtures

2021 ◽  
Vol 12 ◽  
Author(s):  
Adam C. Faller ◽  
Dhivya Shanmughanandhan ◽  
Subramanyam Ragupathy ◽  
Yanjun Zhang ◽  
Zhengfei Lu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Glycine Max ◽  
Pisum Sativum ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Powder Mixtures ◽  
Protein Sources ◽  
Polymerase Chain

Several botanicals have been traditionally used as protein sources, including the leguminous Pisum sativum L. and Glycine max (L.) Merr. While a rich history exists of cultivating these plants for their whole, protein-rich grain, modern use as powdered supplements present a new challenge in material authentication. The absence of clear morphological identifiers of an intact plant and the existence of long, complex supply chains behoove industry to create quick, reliable analytical tools to identify the botanical source of a protein product (many of which contain multiple sources). The utility of molecular tools for plant-based protein powder authentication is gaining traction, but few validated tools exist. Multiplex quantitative polymerase chain reaction (qPCR) can provide an economical means by which sources can be identified and relative proportions quantified. We followed established guidelines for the design, optimization, and validation of qPCR assay, and developed a triplex qPCR assay that can amplify and quantify pea and soy DNA targets, normalized by a calibrator. The assay was evaluated for analytical specificity, analytical sensitivity, efficiency, precision, dynamic range, repeatability, and reproducibility. We tested the quantitative ability of the assay using pea and soy DNA mixtures, finding exceptional quantitative linearity for both targets – 0.9983 (p < 0.0001) for soy and 0.9915 (p < 0.0001) for pea. Ratios based on mass of protein powder were also tested, resulting in non-linear patterns in data that suggested the requirement of further sample preparation optimization or algorithmic correction. Variation in fragment size within different lots of commercial protein powder samples was also analyzed, revealing low SD among lots. Ultimately, this study demonstrated the utility of qPCR in the context of protein powder mixtures and highlighted key considerations to take into account for commercial implementation.

scholarly journals Development of a Quantitative Polymerase Chain Reaction Detection Protocol for Cercospora kikuchii in Soybean Leaves and Its Use for Documenting Latent Infection as Affected by Fungicide Applications

2014 ◽  
Vol 104 (10) ◽  
pp. 1118-1124 ◽  
Author(s):  
A. K. Chanda ◽  
N. A. Ward ◽  
C. L. Robertson ◽  
Z.-Y. Chen ◽  
R. W. Schneider
Keyword(s):  
Polymerase Chain Reaction ◽  
Latent Infection ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Growth Stages ◽  
Symptom Expression ◽  
Chain Reaction ◽  
Cercospora Kikuchii ◽  
Polymerase Chain ◽  
Soybean Leaves

Cercospora leaf blight (CLB) of soybean, caused by Cercospora kikuchii, is a serious disease in the southern United States. A sensitive TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to specifically detect and quantify C. kikuchii in naturally infected soybean plants. The sensitivity was 1 pg of genomic DNA, which was equivalent to about 34 copies of genome of C. kikuchii. Using this qPCR assay, we documented a very long latent infection period for C. kikuchii in soybean leaves beginning at the V3 growth stage (as early as 22 days after planting). The levels of biomass of C. kikuchii remained low until R1, and a rapid increase was detected from the R2/R3 to R4/R5 growth stages shortly before the appearance of symptoms at R6. The efficacy of various fungicide regimens under field conditions also was evaluated over a 3-year period using this qPCR method. Our results showed that multiple fungicide applications beginning at R1 until late reproductive stages suppressed the development of C. kikuchii in leaves and delayed symptom expression. Different fungicide chemistries also had differential effects on the amount of latent infection and symptom expression during late reproductive growth stages.

scholarly journals Assessment ofFusariuminfection in wheat heads using a quantitative polymerase chain reaction (qPCR) assay

2007 ◽  
Vol 24 (10) ◽  
pp. 1121-1130 ◽  
Author(s):  
V. Rossi ◽  
V. Terzi ◽  
F. Moggi ◽  
C. Morcia ◽  
P. Faccioli ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Peptide Nucleic Acid (PNA)-Enhanced Specificity of a Dual-Target Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Assay for the Detection and Differentiation of SARS-CoV-2 from Related Viruses

Diagnostics ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 775
Author(s):  
Won-Suk Choi ◽  
Ju Hwan Jeong ◽  
Halcyon Dawn G. Nicolas ◽  
Sol Oh ◽  
Khristine Joy C. Antigua ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Real Time ◽  
Peptide Nucleic Acid ◽  
Detection Method ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Dual Target

The threat posed by coronaviruses to human health has necessitated the development of a highly specific and sensitive viral detection method that could differentiate between the currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related coronaviruses (SARSr-CoVs). In this study, we developed a peptide nucleic acid (PNA)-based real-time quantitative polymerase chain reaction (RT-qPCR) assay targeting the N gene to efficiently discriminate SARS-CoV-2 from other SARSr-CoVs in human clinical samples. Without compromising the sensitivity, this method significantly enhanced the specificity of SARS-CoV-2 detection by 100-fold as compared to conventional RT-qPCR. In addition, we designed an RT-qPCR method for the sensitive and universal detection of ORF3ab-E genes of SARSr-CoV with a limit of detection (LOD) of 3.3 RNA copies per microliter. Thus, the developed assay serves as a confirmative dual-target detection method. Our PNA-mediated dual-target RT-qPCR assay can detect clinical SARS-CoV-2 samples in the range of 18.10–35.19 Ct values with an 82.6–100% detection rate. Furthermore, our assay showed no cross-reactions with other coronaviruses such as human coronaviruses (229E, NL63, and OC43) and Middle East respiratory syndrome coronavirus, influenza viruses (Type B, H1N1, H3N2, HPAI H5Nx, and H7N9), and other respiratory disease-causing viruses (MPV, RSV A, RSV B, PIV, AdV, and HRV). We, thus, developed a PNA-based RT-qPCR assay that differentiates emerging pathogens such as SARS-CoV-2 from closely related viruses such as SARSr-CoV and allows diagnosis of infections related to already identified or new coronavirus strains.

scholarly journals Quantification of Botrytis cinerea in Grapevine Bunch Trash by Real-Time PCR

2019 ◽  
Vol 109 (7) ◽  
pp. 1312-1319 ◽  
Author(s):  
Melissa Si Ammour ◽  
Giorgia Fedele ◽  
Caterina Morcia ◽  
Valeria Terzi ◽  
Vittorio Rossi
Keyword(s):  
Polymerase Chain Reaction ◽  
Botrytis Cinerea ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Chain Reaction ◽  
Qpcr Method ◽  
Polymerase Chain ◽  
Bunch Rot ◽  
Reliable Quantification

Quantification of colonization of grape bunch trash by Botrytis cinerea is crucial for Botrytis bunch rot (BBR) control. A previously developed quantitative polymerase chain reaction (qPCR) method was adapted to quantify B. cinerea DNA in grape bunch trash, and a colonization coefficient (CC) was calculated as the ratio between the DNA concentrations of B. cinerea and of Vitis vinifera. CC values increased linearly with the number of conidia of B. cinerea or the quantity of mycelium of B. cinerea added to the bunch trash increased. CC values also increased linearly in bunch trash samples containing increasing percentages of B. cinerea-colonized bunch trash; in the latter samples, CC values were correlated with subsequent assessments of B. cinerea colonization of trash (as determined by plating on agar) and sporulation on the trash (as determined by spore counts after incubation in humid chambers). The qPCR assay was also validated using trash collected from bunches treated or not treated with fungicides in three vineyards in two seasons. CC values reflected the reduction in sporulation and in latent infections of mature berries caused by fungicide application. The qPCR assay enables rapid, specific, sensitive, and reliable quantification of the degree of colonization of bunch trash by B. cinerea, which makes it a useful tool for studies of the epidemiology and management of BBR.

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